### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON , 

### D.C

. 20460



### MEMORAN D UM

Date: 6/30/2015 OF FICE OF C HEMICA L SAFET Y AND PO LL U T ION P REVENTION



### SUBJECT: EPTC: Data Eval u ation Reco rd s ( D E R s) for E D S P T i e r 1 

### Ass a ys

PC Code: 041401 

### Decision No.: 459078, 466072 

### Petition No.: NA 

### DP Bar c od e: D397177
, D402841 

### R eg i s tration No .: NA 

### R eg ul a to ry Act i o n : NA 

### Risk Assessment Type: NA 

### TXRNo.: 0051762 

### MRID No.: See Table



### FROM: Greg Akerman, Ph.D. Immediate Office Health Effec ts Division (7509P) 

### Case N o. : NA 

### CAS No. : 759-94-4 

### 40 CF R: NA



### THROUGH: Jess Rowland

## de-~~ Deput y Director Health Effec ts Division



### TO: Jolene Trujillo Biologist / Chemical Review Manager Risk Management and Implementation Branch V Pesticide Re-evaluation Division (7505P)

I. 

### ACTION REQUESTED Ver.Apr. 20 I 0



### The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays submitted in response to the agency ' s Test Order for EPTC: Test Order # EDSP-041401-64.

Pa ge I of2Page 2 of 2 

### II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for EPTC.

III. MRID Table

Chemical: EPTC PC Code: 041401 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48827601 890.1150 Androgen Receptor Binding (Rat Prostate) 48616403, 48843501 890.1200 Aromatase Assay (Human Recombinant) 48616402 890.1250 Estrogen Receptor Binding 48616404, 48843501 890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48616405

890.1350 Fish Short-Term Reproduction 48827602 890.1400 Hershberger (Rat) 48616406 890.1450 Female Pubertal (Rat) 48827603 890.1500 Male Pubertal (Rat) 48827603 890.1550 Steroidogenesis (Human Cell Line – H295R) 48816401 890.1600 Uterotrophic (Rat) 48616407Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 1 of 80

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 402838

OECD Data Point 231

EPA MRID 48827601

EPA Guideline 890.1100 Amphibian Metamorphosis Assay (Frog)

Test Material: EPTC Purity (%): >99.0%

Common Name EPTC
Chemical Name IUPAC Not reported
CAS Name Not reported

CAS No. 759-94-4 Synonyms Eptam® Technical (EPTC); S-Ethyl dipropylthiocarbamate

EPA PC Code 041401

Primary Reviewer: John Marton Date: 9/24/12 Staff Scientist, Cambridge Environmental, Inc.

Secondary Reviewer(s): Teri S. Myers Date: 9/27/12 Senior Scientist, Cambridge Environmental, Inc.

Reviewer: Frank T. Farruggia, Ph.D. Date: 3/22/13

USEPA/OPP/EFED/ERB3

Reviewer: Robin Sternberg Date: 11/1/13 Wildlife Biologist, USEPA/OPP/EFED/ERB1

Additional Reviewer: Amy Blankinship Date: 6/5/15

USEPA/OPP/EFED/ERB6 

## FRANK FARRUGGIA Digitally signed by FRANK FARRUGGIA DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=FRANK FARRUGGIA, dnQualifier=0000048865 Date: 2015.06.11 08:18:40 -04'00'

Digitally signed by ROBIN STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126 Date: 2015.06.11 11:19:25 -04'00'



## AMY BLANKINS HIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.15 17:33:27 -04'00'Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 Date Evaluation Completed: 6/5/15

CITATION: Lee, Michael R. 2012. EPTC- Amphibian Metamorphosis Assay with African Clawed Frog ( Xenopus

laevis ) Following OPPTS Test Guideline 890.1100 and OECD Test Guideline No. 231. Unpublished study
performed by Smithers Viscient, Wareham, Massachusetts, 02571-1037. Laboratory report number 12791.6155. Study sponsored by Gowan Company, Yuma, Arizona. Study completed May 4, 2012.

Note: The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to
identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across
the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determi ne if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to aid
in preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test Guidelines, nor to provide any guidance on how the study should be conducted.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of EPTC (>99.0% purity) on amphibian metamorphosis of the African clawed frog ( Xenopus

laevis ) was conducted under flow-through conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80 tadpoles per control or treatment, 20/replicate) were exposed to nominal concentrations of 0 (negative control), 0.024, 0.12, 0. 60, and 3.0 mg a.i./L. Mean-measured concentrations were <0.0063 (<LOQ; control), 0.019, 0.10, 0.50, and 2.6 mg a.i./L. The test system was maintained at 21 to 23°C and a pH of 7.0

to 8.2.

There were no treatment-related effects on survival of tadpoles during the 21-day test: survival was 100% in the
negative control and 98, 99, 100, and 96% in the mean -measured low, mid-low, mid-high, and high treatment groups. At test termination, spinal curvature was observed in 33% of tadpoles in the negative control and 41, 44, 28, and 0% in the mean-measured low, mid-low, mid-high, and high treatment groups. The incidence of spinal

curvature was not considered to be treatment-related.

At the three highest test concentrations, day 7 snout-vent length (SVL) was significantly reduced (p<0.05) by 6.7, 7.8, and 15.2%, respectively, and day 7 body weight was significantly reduced (p<0.05) by 16.1, 22.2, and
41.9%, respectively, compared to the negative control. At the three highest test concentrations, day 21 body weight was significantly reduced (p<0.05) by 5.9, 12.0, and 22.9 %, respectively, compared to the negative control. Day 21 SVL was significantly reduced (p<0.05) at all treatment concentrations compared to the negative control (range: 2.2-8.5%).

EPTC significantly decreased (p<0.05) day 21 normalized hind-limb length (HLL) by 17.5% at the high treatment level compared to the negative control; no inhibitions (p >0.05) were observed in Day 7 normalized HLL. There
was no significant acceleration or delay of median NF developmental stage at any treatment level throughout the
exposure period. No asynchronous development was observed.

Mild follicular cell hyperplasia and hypertrophy were ob served in the negative control and all treatment groups,

and one to two individuals with increased follicular lume n area (mild) were observed in each of the negative controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 4 of 80

DER Template Version: 22 September 2011 and mid-low and high treatment groups. However, these histopathology findings were not considered to be

treatment-related.

The study met all of the performance and validity criteria.

The assay satisfies the EDSP Tier 1 Test Order requirements for an Amphibian Metamorphosis Assay (OCSPP Guideline 890.1100).

Results Synopsis:

Test organism NF stage at test initiation: NF 51
Test organism total length at test initiation (optional): Not reported
Test type: flow-throughData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 in the Amphibian Metamorphosis Assay (AMA) with EPTC.

Treatment
(mg a.i./L)

[measured] NF Developmental Stage Hind Limb
Length 3 Asynchronous
Development Thyroid Gross

and
Histopathology
Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21

0.019 No No No No No No No

0.10 No No No No No No No

0.50 No No No No No No No

2.6 No No No Yes No No No

1 A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless

otherwise specified.

2 The criteria for significance are described in the Review er’s Analysis and Statistical Ve rification sections of the

DER. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified
pathologist.

3 Hind-limb length is normalized to snout-vent length (SVL).

I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in the U.S. Environmental Protection Agency’s Endocrine Disruptor Screening Program Test Guidelines 890.1100, Amphibian Metamorphosis (Frog), and OECD Guideline for Testing
of Chemicals No. 231, Amphibian Metamorphosis Assay. The following

deviations from OCSPP 890.1100 were noted:

1. The flow rate resulted in a 90% replacement time in the test vessels of approximately 8 hours. OCSPP
guideline 890.1100 recommends complete volume turn over every 2.7 hours. The flow-through rate of 32.5 mg/min as calculated by the reviewer is greater than recommended ( i.e. , 25 mL/min).Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 2. Results from the periodic screening analysis of the dilution water were not provided.

These deviations do not impact the acceptability of the study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements
were provided. This study was conducted in compliance with all pertinent OECD (OECD, 1998) and U.S. EPA (40 CFR, Part 160 and 40 CFR, Part 792) Good
Laboratory Practice regulations with th e following exception: routine food and water contaminant screening analyses were conducted using standard U.S. EPA procedures by GeoLabs, Inc., Braintree, Massacusetts.

A. Test Material EPTC

Description: Liquid
OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or Kow,
vapor pressure of test compound, expiration date.

Lot No./Batch No. : P1105096 (Lot No.)
Purity: >99.9%
Impurities: Not reported Stability of Compound: Stable. Mean-measured concentrations over the 21-day exposure period yielded recoveries of 78-87% of nominal with coefficients of variation of 3.5

to 6.8%. Storage Conditions of

Test Chemicals: Stored at room temperature in a dark, ventilated cabinetData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page7of80 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: African clawed frog EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be used as an alternate species1 ; however, a list of all of the necessary protocol deviations to accommodate this spec ies is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be

identified.Species scientific name: Xenopus laevisSpecies strain (if stated): Not specified1U.S. Environmental Protection Agency (EPA). (2011). Correctionsand Clarifications on Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington,D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page8of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained asin-house stock?Yes The brood stock was originally obtainedfrom Nasco, Fort Atkinson, Wisconsin andwas maintained as in-house breeders forapproximately one year prior to use forgenerating tadpoles used in this exposure. EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?YesAcclimation period for parentalfrogs (if applicable):At least one yearDetails on parental feeding: Not specifiedDuring the pre-exposure period, tadpoleswere fed twice daily increasing from 5 to30 mg/tadpole.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page9of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on parental health: The brood stock did notshow any signs ofdisease, sickness,injuries, or abnormalitiesform the day of receipt tothe day of pre-exposureinitiation.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page10of80 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?Yes EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn

(i.e., the spawns are not co-mixed)Number of spawns evaluated (ifapplicable):3, one from each ofthree breeding pairsFrom hatch to feeding stage(approximately 6 days post-fertilization),the quality of each of the three spawnswas quantitatively assessed; criteriawere embryo survival and the quantityof embryos.Number of eggs sampled perspawn:Not specifiedEach spawn produced at least 1,500 eggsOnce all tadpoles were at feeding stage(day 6 post-fertilization), larvae from thehighest quality spawn were transferred tonine, 10-L rearing tanks. All transferredtadpoles originated from a single spawn.NF stage at test initiation 51 EPA recommends that the definitive study be initiated with larvae at Nieuwkoop – Faber (NF) developmental stage 51 ( 17 days post-fertilization).Age at test initiation:12 days post-fertilization(dpf)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page11of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMean total length at test initiation(if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedWas the optional size selectionmethod used?NoDetails on larval selection: Healthy tadpoles at NFstage 51 were selected.No further details wereprovided.Loading rate (rearing density): 10 larvae/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for flow-through systems or 4 tadpoles/L in static-renewal exposure systems.Type of food:Xenopus Expresstadpole foodRepresentative samples of the foodsource were periodically analyzed for the EPA recommends Sera Micron ® throughout pre-exposure (after NF stage 45/46) andData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page12of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationspresence of pesticides, PCBs and toxicmetals by GeoLabs, Inc., Braintree,Massachusetts. None of thesecompounds have been detected in thefood samples at concentrationsconsidered toxic to the test organisms.Based on these analyses, food sourceswere considered to be of acceptablequality since all analyte concentrationswere below levels of concern, inagreement with ASTM (2005). during the entire 21-d definitive study. If another diet is used, the study report should provide analysis of iodide content and potential contaminants, and the diet should demonstrate equal performance to Sera

Micron ® .Source of food:Xenopus Express,Brooksville, FloridaIodide concentration in diet (ifknown):Not reportedFrequency of feeding:2 times/day EPA recommends that feeding occur at least twice per day.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page13of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding regime: Feeding regime followedguidelinerecommendations. It is recommended that food rations during the pre-exposure period be increased along with larval growth to approximately 30 mg/larva/day by test initiation. EPA and OECD recommend that food rations increase from 30 mg/larva/day at test initiation (Study Day 0-4) to 80 mg/larva/day in the last week of the test (Study Day 15-21).Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page14of80 DER Template Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through

system.Type of flow-through dilutionsystem (if applicable):Intermittent-flow proportionaldiluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):32.5 mL/min(calculated by reviewer)The flow rate providedapproximately 7.2 volume additionsevery 24-hours, with a 90%replacement time of ~8 hours.(6.5 L*7.2*1000 mL/L)/(24 hr *60 min/hr) Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page15of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):Test material was delivered to amixing chamber using a syringepump and a gas-tight syringe. Themixing chamber was positionedover a magnetic stir plate whichaided in mixing the stock solutionwith the dilution water. The solutionin the mixing chamber constitutedthe highest nominal testconcentration (3.0 mg a.i./L) andwas subsequently proportionallydiluted (20%) to provide theremaining nominal exposureconcentrations (0.60, 0.12, and0.024 mg a.i./L).Flow-splitting cells were employedto equally distribute the solutions tothe four replicate vessels for eachconcentration or control group.Flow-splitting accuracy of the dilutercells was within ±5% of the nominalvalue. Recommended toxicant mixing for flow-through systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing;3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is
within 10%.Renewal period for staticrenewal (if applicable):NA If static renewal is used, EPA recommends 24-hr renewal; renewal period is recommended not to exceed 72 hours.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page16of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAeration? Nonereported EPA recommends maintaining dissolved oxygen concentrations >40% air saturation (>3.5 mg/L). Aeration may be maintained through bubblers. It is recommended to set bubblers at levels that do not cause stress on the tadpoles.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page17of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water: FETAX solution was used asdilution water. Laboratory wellwater was fortified according to theFETAX formulation in “StandardGuide for Conducting the FrogEmbryo TeratogenesisAssay-Xenopus (FETAX)”(ASTM, 2004).The laboratory well water used toprepare FETAX solution originatedfrom an on-site 100-meterbedrock well. The water waspumped to a concrete reservoirwhere it was supplemented ondemand with de-chlorinated Townof Wareham well water. The waterwas aerated prior to use to ensureproper equilibration of dissolvedgases.Solutions were prepared in 3,400-Lbatches and mixed for approximately48 hours prior to use to ensure allsalts were properly dissolved andthat dissolved gases were atequilibrium with the atmosphere. EPA recommends natural or reconstituted water; it is recommende d that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants, including known substrates of the iodine transporter of the thyroid gland (e.g., fluoride, chlorate, perchlorate). OECD accepts any water in which the test species show control survival at least as good as indicated in the test

guideline.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page18of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzed forpesticides, heavy metals, andother contaminants?Yes. No pesticides, PCBs, or toxicmetals were detected atconcentrations considered toxic inany of the water samples analyzed,in agreement with ASTM (2005)standard practices.Iodide supplementation inwater?None reported If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 ս g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).Test vessel type/materials: Glass EPA and OECD recommend that water-contact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page19of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel size:30 x 15 x 20 cm2.5 gallon exposure aquaria14.5-cm high side drainFill volume:~6.5 LAdditional details on exposuresystem:The exposure system wasconstructed of glass, siliconesealant, stainless steel, andTeflon®. All test chambers werebrushed and siphoned daily toremove detritus and uneaten foodduring the test. Calibration of thediluter system was conducted priorto exposure initiation andconfirmed at test termination (Day21) by measuring delivery volumesof toxicant and dilution water.The function of the diluter wasmonitored daily and a visual checkwas performed twice daily. Inaddition, weekly analysis ofexposure solutions for testsubstance concentration was alsoused to verify proper operation of thediluter system.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page20of80 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsHardness (mg/L as CaCO3) 160 1801701Measured in replicate A ofthe control, low, and hightest concentrations on Day0 and in alternatingreplicates weekly. EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH 7.018.167.74Measured in all testvessels on Day 0 and dailyin alternating replicates.Temperature wascontinuouslymonitored in replicate A ofthe control EPA recommends pH 7.5 ± 1, inter-replicate and inter-treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L) 3.97 9.236.97 EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L (>40% air
saturation).Temperature (°C)21.4 22.721.9 EPA recommends temperature

22±1 o C; inter-replicate and inter-treatment differentials should not exceed 0.5 o C.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page21of80 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsIodide (սg/L) NR NR 7.8Well water (January 2012) EPA recommends aquatic iodide range 0.5 – 10 ս g/L (supplemental iodide should not exceed 2 ս g/L).Ammonia Notdetermined General recommendations for frequency of measurements: EPA recommends that water quality parameters be measured in a control and at one test item concentration at least weekly. In static renewal systems, water quality parameters, including ammonia, should be measured just prior to renewal. In addition, EPA recommends that DO be measured at each concentration at least weekly and that temperature be measured continuously. OECD recommends that DO and temperature be measured at leastFluoride NotdeterminedPerchlorate NotdeterminedChlorate NotdeterminedAlkalinity (mg/L as CaCO3)Conductivity (սS/cm)80170092190086118001Measured in replicate A ofthe control, low, and hightest concentrations on Day0 and in alternatingreplicates weekly.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page22of80 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurement IntervalGuideline Recommendations weekly and that pH and hardness be measured at least at the beginning and end of the test.1Calculated by reviewer at average of minimum and maximum.D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?YesIf yes, what was the method fordetermining the highest testconcentration in therange-finder?Expected toxicityThe doses for preliminary acutetesting were based on the published fishacute toxicity information of EPTC (LC50values of 14 and 18 ppm for trout andbluegill, respectively), the solubility limit ofEPTC in water (370 mg/L at 20 °C), andconsultation with the Study Sponsor. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., <10% mortality), whichever concentration is lowest.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page23of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies: Xenopus laevisLife stage:NF stage 51Test duration:4 daysAdditional details:After 96 hours of exposure, theLC50and EC50values were 18(95% C.I.: 16-21) and 9.1 (95%C.I.: 7.7-11) mg a.i./L,respectively, based on nominalconcentrations. There was nomortality at13 mg a.i./L and100% mortality at 25 and 50 mga.i./L. At 13 mg a.i./L, alltadpoles exhibited adverseeffects ( i.e., pale color, lethargy,or loss of equilibrium) at 96hours.Since the analytical recoveriesTwo initial preliminary 96-hour acuterange-finding studies were conducted understatic-renewal conditions (renewal at 48hours) at 1.6, 3.1, 6.3, 13, 25, and 50 mga.i./L and 1.6, 3.1, 6.3, 13, and 25 mg a.i./L,respectively.Due to lower than expected measured EPTCconcentrations and suspected volatility anddegradation of the test substance in the dilutionmatrix, a third range-finding exposure wasperformed using nominal concentrations of 1.6,3.1, 6.3, 13, 25 and 50 mg a.i./L underflow-through conditions.The final range-finding exposure wasconducted using an exposure systemData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page24of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsduring the preliminary acuteexposure were slightly variable,theEC50 calculation was based onnominal dose.Water quality parametersmeasured during the 96-hourexposure period established adissolvedoxygen range of 7.8 to 8.6mg/L, a pH range of 8.2 to 8.3,and a temperature range of 21to 22 °C.consisting of an intermittent-flowproportional diluter, a temperaturecontrolled water bath, and a set ofeight exposure aquaria with a total solutionvolume of 6.5 L. Two replicates weremaintained for each concentration andcontrol. Each replicate vessel contained 20tadpoles at exposure initiation. Testorganisms were monitored daily forobservations of mortality and abnormalbehavior. During the exposure, dissolvedoxygen, pH and temperature weremonitored daily. Samples from eachreplicate of each concentration weresampled at 24 hours and again at the72-hour interval from replicate A of eachconcentration to confirm exposure dose.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page25of80 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be

21 days.Method for selecting the highesttest concentration in the definitivetest:Range-finder; fishshort-term reproductiveassay with EPTC andconsultation with theStudy Sponsor. Highestnominal testconcentration ( i.e., 3.0mg a.i./L) wasapproximately 1/3 of thenominal EC50value (9.1mg a.i./L). EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., <10% mortality), whichever concentration is

lowest.Reference study citation (ifapplicable):NAData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page26of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSeparation of test concentrations:0.2 Typically for these exposures, a 10% dilution series isapplied with three test substance concentrations.However, based on the results of the fish short-termreproductive assay with EPTC, the dilution series wasincreased to 20% and a fourth concentration wasincluded. For the fish study, the high dose was set using1/3 of the 96-hour LC50 for adult fish, threeEPTC-dosed concentrations separated by a dilutionfactor of 10%. During this exposure, latent lethal andsublethal responses (approximately exposure day 11)were observed in the high and mid doses. For theamphibian study, the intent of a 20% dilution series andfour EPTC-dosed treatment doses was to avoid thelatent responses observed in the fish study and stillmaintain > 2 uncompromised EPTC exposureconcentrations. EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Number of test concentrations:4 EPA recommends a minimum of 3 concentrations and a control,Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page27of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ): 0.0062 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD): Not reportedFrequency of measurement: Samples were collectedfor analysis on Days 0,7, 16, and 21.At exposure initiation (day 0), samples from replicateA of each treatment and control solution were removedand analyzed and from alternating replicates weeklythereafter. On exposure day 0, an interfering peak(possibly related to the matrix in an aging test system)was present in the control vessel. On first glance, this It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week
thereafter.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page28of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationsappeared to be control contamination, therefore allcontrol vessels were sampled on day 1. Upon furtherdiagnosis, this interfering peak was found to be presentin all samples. The area of this interfering peakapproximated the area of the low standard and wasimpacting the limit of quantitation (LOQ) for thisanalysis. As a result of this interference, the lowconcentration calibration standard was removed fromthe standard curve and the LOQ for this analysis wassubsequently increased to offset any potential impactfrom this interfering peak. Since the peak observed inthe control vessel at day 0 was not determined to beEPTC, the day 1 sampling was not necessary andtherefore, not reported.Number of replicates in control: 4 EPA recommends 4 replicates.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page29of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in solventcontrol (if applicable):NA EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of larvae per treatment attest initiation:80Was a solvent used?NoSolvent type (if applicable): NAData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page30of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed 0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used? NoPositive control (if applicable): NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12 (light:dark).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series onTesting and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page31of80 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’s surface: 0.750-1.2 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:NAData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page32of80 DER Template Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with EPTC.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)0.00 <0.0062 NA NA EPA and OECD recommend that test item concentrations be maintained at a coefficient of variation (CV) 20%.Solvent control (ifapplicable)NA NA NA NATreatment 10.0240.0195.1NATreatment 20.120.106.5NATreatment3 0.60 0.5013.5 NATreatment 43.02.64.9NAAbbreviations:CVCoefficient of variation.1calculated as 0.49 by study authorData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 E. Observations

Biological Endpoints: Day 7: Development stage, wet weight, snout-vent length (SVL), hind-limb length (HLL), normalized HLL (HLL:SVL) Day 21: Development stage, wet weight, SVL, HLL, normalized HLL, thyroid histopathology (tadpoles from
the mean-measured 0.49 mg a.i./L treatment group were not included in the histopathology analysis)
Daily: Mortality, sub-lethal effects

Were raw (individual) data provided? Yes

EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (days 7 and 21); hind limb length (days 7 and 21); snout-vent length (days 7 and 21); body weight (test initiation, for optional size-based larval selection); and thyroid gland gross pathology and histopathology (day 21). Note the histopathology
section of the test guideline also incl udes thyroid gross pathology observations.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 II. RESULTS AND DISCUSSION

A. Results

By test termination, mean survival was 100% in the negative control and 98, 99, 100, and 96% in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively (Table 9).

Table 9: Larval Mortality in Xenopus laevis

.

Treatment (mg a.i./L)
[measured] Larval Mortality

Day 7 1 Day 21

n Mortality # Mortality % n Mortality # Mortality %

Negative Control 80 NR NR 60 0 0

0.019 80 NR NR 60 2 2

0.10 80 NR NR 60 1 1

0.50 80 NR NR 60 0 0

2.6 80 NR NR 60 3 2 4

Abbreviations: NR=Not reported

1 Sample size and cumulative mortality values at Day 7 prior to interim sacrifice were not reported. Further, only

cumulative Day 21 survival was reported. The study author did not report when mortalities occurred.

2 Two mortalities occurred in one of the replicates.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Median developmental stage on Day 7 was 54 in the negative control and 0.019-0.50 mg a.i./L treatment groups and 53 in the mean-measured 2.6 mg a.i./L treatment group (Table 10). By Day 21, median
development stage was 57 in the negative control and all treatment groups except the mean-measured 0.50 mg a.i./L treatment group where median developmental stage averaged 57.5. The 10 th and 90 th
percentiles in Day 21 developmental stage did not differ by more than four NF stages. No asynchronous
development was reported.

On day 21, there was only 1 (1.7%) tadpole greater than stage 60 ( i.e. , stage 61) in each of the
mean-measured 0.019 and 0.50 mg a.i./L treatment groups. These tadpoles were excluded from analyses with respect to body weight, snout vent length, and hind leg length (including normalized hind leg length) by the study author and reviewer.

Table 10: Larval Development in Xenopus laevis – Developmental Stage and Asynchronous Development.

Treatment (mg a.i./L)
[measured] Developmental Stage

Day 7 Day 21

n Median Stage # Asynchronous n Median Stage # Asynchronous

Negative Control 4 54 0 4 57 0

0.019 4 54 0 4 57 0

0.10 4 54 0 4 57 0

0.50 4 54 0 4 57.5 0

2.6 4 53 0 4 57 0Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Day 7 normalized HLL averaged 0.12 in the negative control and 0.11 in the mean-measured 0.010, 0.10, 0.49, and 2.6 mg a.i./L treatment groups (Table 11). Day 21 normalized HLL averaged 0.44 in the
negative control and 0.45, 0.44, 0.44, and 0.36 in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups (Table 11).

Table 11: Larval Development in Xenopus laevis – Hind Limb Length.

Treatment (mg a.i./L)

[measured] Hind Limb Length (HLL)

Day 7 Day 21 2

n Mean (mm) ±SD HLL:

SVL 1 n Mean (mm) ±SD HLL:

SVL 1

Negative control 4 2.31 0.08 0.12 4 13.64 0.37 0.44

0.019 4 2.07 0.21 0.11 4 13.74 0.71 0.45

0.10 4 2.05 0.18 0.11 4 13.30 0.54 0.44

0.50 4 1.99 0.11 0.11 4 12.90 0.97 0.44

2.6 4 1.81 0.10 0.11 4 10.39 0.44 0.36

Abbreviations: NA Not applicable. SD Standard deviation.

1 Summary results for snout-vent length (SVL) are presented in the next table ( Table 12 ).

2 Day 21 HLL:SVL values are the median values reported by the study author.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Mean Day 7 SVL averaged 20.13 mm in the negative control and 18.95, 18.78, 18.55, and 17.08 mm in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively (Table 12).
Mean Day 21 SVL averaged 31.15 mm in the negative control and 30.45, 30.15, 29.56, and 28.50 mm
in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively.

Mean Day 7 body weight averaged 0.57 g in the negative control and 0.50, 0.48, 0.45, and 0.33 g in the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively (Table 12).
Mean Day 21 body weight averaged 1.92 g in the negative control and 1.89, 1.80, 1.69, and 1.48 g in
the mean-measured 0.019, 0.10, 0.50, and 2.6 mg a.i./L treatment groups, respectively.

Table 12: Larval Growth in Xenopus laevis

.

Treatment (mg a.i./L)
[measured] Snout-Vent Length (SVL) 2 Body Weight 1

Day 7 Day 21 Day 7 Day 21

n Mean (mm) ±SD n Mean (mm) ±SD n Mean (g) ±SD n Mean (g) ±SD

Negative

Control 4 20.13 0.19 4 31.15 0.68 4 0.57 0.07 4 1.92 0.11

0.019 4 18.95 1.03 4 30.45 0.24 4 0.50 0.07 4 1.89 0.02

0.10 4 18.78 0.67 4 30.15 0.52 4 0.48 0.04 4 1.80 0.07

0.50 4 18.55 0.66 4 29.56 0.41 4 0.45 0.04 4 1.69 0.08

2.6 4 17.08 0.78 4 28.50 0.35 4 0.33 0.06 4 1.48 0.05

Abbreviations: SD Standard deviation.

1 Also referred to as “wet weight” in the test guideline.

2 SVL values represent the mean of the replicate mediansData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Tadpoles selected for histological analysis were stage-matched to the median of the control (stage 57),
if possible. If there were replicates with more than five larvae at the appropriate stage, then five larvae were randomly selected. If there was not a sufficient number of tadpo les at the appropriate stage, the next stage up was selected (stage 58), until a total of five tadpoles were selected. If an insufficient number
of tadpoles still remained, the next stage down from the median of the control was selected until a
total of five tadpoles were selected.

No samples from the mean-measured 0.50 mg a.i./L treatment group were analyzed for histopathological

effects.

The following lesions were not present in any examined sections from study tadpoles: thyroid gland hypertrophy, thyroid gland atrophy, decreased follicular lumen area, colloid depletion, changes in colloid quality, and metamorphic pharyngeal changes. Mild fo llicular cell hypertrophy and hyperplasia were
observed in the control and all treatment groups. Mi ld follicular lumen area increase was observed in
the negative control 0.1 and 2.6 mg a.i./L treatment groups. The similarity in incidence and mild severity across groups indicates that these were not treatment-related findings, but a reflection of normal developmental variation (Tables 13 and 14).Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis

.

Treatment
(mg a.i./L)
[measured] Diagnostic Observations 1

Severity Thyroid Gland
Hypertrophy Thyroid Gland
Atrophy Follicular Cell
Hypertrophy Follicular Cell
Hyperplasia

n Incidence n Incidence n Incidence n Incidence
Negative Control 0 20 20 20 20 20 17 20 18

1 20 0 20 0 20 3 20 2

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.019 2 0 19 19 19 19 19 17 19 15

1 19 0 19 0 19 2 19 4

2 19 0 19 0 19 0 19 0

3 19 0 19 0 19 0 19 0

0.10 0 20 20 20 20 20 19 20 18

1 20 0 20 0 20 1 20 2

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.50 3 0 NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA

2 NA NA NA NA NA NA NA NA

3 NA NA NA NA NA NA NA NA

2.6 0 20 20 20 20 20 18 20 17

1 20 0 20 0 20 2 20 3

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

1 Thyroid gland gross pathology and histopathology are graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild,

2=Moderate, 3=Severe. See OECD No. 82 for reference.

2 One sample was missing from this group (n = 19).

3 Samples were not analyzed from this group.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page40of80 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment(mg a.i./L)[measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control 0 20 19 20 20 NA NANA NA NA NA1 20 1 20 0 NA NANA NA NA NA2 20 0 20 0 NA NANA NA NA NA3 20 0 20 0 NA NANA NA NA NA0.019 0 19 19 19 19 NA NANA NA NA NA1 19 0 19 0 NA NANA NA NA NA2 19 0 19 0 NA NANA NA NA NA3 19 0 19 0 NA NANA NA NA NA0.10 0 20 18 20 20 NA NANA NA NA NA1 20 2 20 0 NA NANA NA NA NA2 20 0 20 0 NA NANA NA NA NA3 20 0 20 0 NA NANA NA NA NA0.50 0 NANA NA NA NA NANA NA NA NA1 NANA NA NA NA NANA NA NA NAData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page41of80 DER Template Version: 22 September 2011Treatment(mg a.i./L)[measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence2 NANA NA NA NA NANA NA NA NA3 NANA NA NA NA NANA NA NA NA2.6 0 20 19 20 20 NA NANA NA NA NA1 20 1 20 0 NA NANA NA NA NA2 20 0 20 0 NA NANA NA NA NA3 20 0 20 0 NA NANA NA NA NA1Thyroid gland gross pathology and histopathology are graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference.2One sample was missing from this group (n = 19).3Samples were not analyzed from this group.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Beginning on Day 13, several tadpoles in the negative control and most treatment groups were observed with spinal curvature. By test termination, spinal deformities were observed for 33% of negative control tadpoles and 41, 44, 29, and 0% of tadpoles in the mean-measured 0.019, 0.10, 0.49, and 2.6 mg a.i./L treatment groups, respectively (Table 15). These observations were not considered to be
treatment-related. For studies conducted at Smithers Viscient, there is no correlation between the presence of spinal curvature and the increase in % CV for potentially affected endpoints (e.g., SVL, HLL, wet weight).

Table 15: Clinical Signs in Xenopus laevis

.

Treatment
(mg a.i./L)

[measured] Clinical Signs 1

Type n Incidence at Test Termination (%)

Negative Control Spinal curvature 60 33

0.019 Spinal curvature 58 41

0.10 Spinal curvature 59 44

0.50 Spinal curvature 60 28
2.6 Spinal curvature 57 0

1 Note that asynchronous development (unable to stage) is reported previously in Table 10

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DER Template Version: 22 September 2011 B. Study Author’s Analysis and Conclusions

Developmental stage, SVL, HLL, and wet weight were statistically analyzed to identify significant
reductions in the treatment organisms compared to control organisms. All statistical conclusions were made at the 95% level of certainty except in the case of the basic assumption tests (e.g., Shapiro-Wilks’ Test and Bartlett’s Test in which the 99% level of certainty was applied). When necessary, the OECD
Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application was consulted (OECD, 2006). CETIS-Comprehensive Environmental Toxicity Information System TM (Ives, 2011) Version 1.8.4.18 software was used to perform all statistical computations.

Mortality was analyzed using step-down Cochran-Armitage test (step down) for monotonic trend or Fisher's Exact test with Bonferroni-Holm adjustment for non-monotonic trend.

Developmental stage was analyzed using the Jonckheere-Terpstra test (step-down). When monotonic
trends were present, HLL, SVL, and wet weight were analyzed using the Jonckheere-Terpstra test (step-down). When trends were non-monotonic, data were tested for normality and homogeneity of variance. If the data met the assumptions of parametric statistics, Dunnett’s Multiple Comparison Test
or the Tamhane-Dunnett tests were used. If the data did not meet these assumptions, the Mann-Whitney test with Bonferonni-Holm adjustment was employed.

For larval survival, Fisher's Exact Test with Bonf erroni-Holm Adjustment determined no significant difference in any treatment level tested compared to the control.

Day 7 data
The day 7 developmental stage was analyzed by applying the multi-quantal Jonckheere Test to the 20th through the 80th percentiles for all treatment levels compared to the control. The multi-quantal procedure determined a significant reduction in the day 7 developmental stage among tadpoles exposed to the 2.6
mg a.i./L treatment level compared to the control. Jonckheere-Terpstra Step-Down Test determined a significant difference in day 7 whole body wet weight
and snout-vent length among tadpoles exposed to all treatment levels tested compared to the control.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Jonckheere-Terpstra Step-Down Test determined a signi ficant reduction in day 7 hind limb length among
tadpoles exposed to the 0.49 and 2.6 mg a.i./L treatment levels compared to the control. Dunnett's Multiple Comparison Test determined a significant reduction in day 7 hind limb length
normalized by SVL among tadpoles exposed to the 2.6 mg a.i./L treatment level compared to the control.

Day 21 data
The day 21 developmental stage was analyzed by applying the multi-quantal Jonckheere Test to the 20th through the 80th percentiles for all treatment levels compared to the control. The multi-quantal procedure determined no significant reductions in the day 21 developmental stage among tadpoles exposed to any
of the treatment levels tested compared to the control. Jonckheere-Terpstra Step-Down Test determined a significant difference in day 21 whole body wet weight
and hind limb length among tadpoles exposed to treatment levels > 0.10 mg a.i./L compared to the

control. Jonckheere-Terpstra Step-Down Test determined a significant difference in day 21 snout-vent length
among tadpoles exposed to all treatment levels tested compared to the control. Jonckheere-Terpstra Step-Down Test determined a significant reduction in day 21 hind limb length normalized by SVL among tadpoles exposed to the 2.6 mg a.i./L treatment level compared to the control.

In summary, on Day 7, both developmental stage and normalized HLL were significantly reduced at the highest concentration relative to the negative control. Further, median wet weight and median SVL were significantly inhibited on Day 7 at all treatment levels relative to the negative control, while median HLL was significantly reduced at the two highest treatment groups.

Similar to Day 7, median normalized HLL at Day 21 was significantly reduced at the highest treatment
level relative to the negative control. However, developmental stage at Day 21 was comparable between the negative control and all treatment groups. Day 21 wet weight and HLL were significantly reduced at

the mean-measured  0.10 mg a.i./L treatment groups, relative to the negative control, whereas SVL was

reduced at all treatment levels.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 There were no treatment-related histopathological effects, and no asynchronous development was observed. Further, the test material did not appear to have any effect on sub-lethal effects.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: Day 7 wet weight, SVL, and HLL had monotonic trends and were analyzed using the Jonckheere-Terpstra test. Day 7 developmental endpoints ( i.e. , NF stage and normalized HLL) were not monotonic and did not meet the assumptions of normality and homogeneity of variance ( i.e.
, Shapiro-Wilks’ and Bartlett’s Tests, respectively) and were analyzed using the Mann-Whitney Test. Day
21 developmental stage was not monotonic and did not meet the assumptions of parametric statistics and
was analyzed with the Mann-Whitney test. All Day 21 growth parameters had a monotonic response and
were analyzed using the Jonckheere-Terpstra test. Day 21 normalized HLL did not have a monotonic response but met the assumptions of parametric statistics and was therefore analyzed with Dunnett’s test.
All analyses were conducted using SAS version 8.1. There were only two individuals from this study (0.024 mg a.i./L, Tank C and 0.60 mg a.i./L, Tank A) exhibiting developmental stages >60; and both
were at the 61 stage by day 21. Replicate means and medians for body weight, snout vent length, and hind leg length (including normalized hind leg length) were calculated excluding all tadpoles at NF stage

 61.

Conclusions:

On Day 7, the reviewer did not find significant inhibitions in developmental stage or normalized HLL ( p < 0.05). However, all growth parameters ( i.e. , SVL, body weight, and HLL) were significantly reduced
in the mean-measured 0.10, 0.50, and 2.6 mg a.i./L treatment groups relative to the negative control ( p

<0.05). On Day 21, there were no significant effects on developmental stage. However, normalized HLL was significantly reduced at the highest treatment level (18%; Dunnett’s, p = 0.002). Day 21 weight
was significantly reduced at the three highest treatm ent groups relative to the negative control (6-23%
reductions; Jonckheere-Terpstra, p  0.020). Day 21 SVL was significantly reduced at all treatment levels (2-9% reductions; Jonckheere-Terpstra, p  0.04). Day 21 HLL, however, was only significantly reduced at the highest treatment level (24% reduction; Jonckheere-Terpstra, p < 0.001).Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page46of80 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with EPTC.Treatment(mg a.i./L)[measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21Median pMedian p%Diff. p% Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)NegativeControl54 NA 57 NA 0 NA 0 NA NANANANANo0.01954 0.48 57 0.48 -5.9 0.13 2.5 0.92NA NA NA NA No0.1054 1.0 57 1.0 -5.2 0.68 0.23 1.0NA NA NA NA No0.5054 1.0 57.5 0.22 -7.4 0.066 -0.23 1.0NA NA NA NA No2.6 53 0.10 57 1.0 -8.5 0.065 -17.50.002NA NA NA NA NoStatistical Test Mann-Whitney Mann-Whitney Mann-Whitney Dunnett’s NANANAData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis AssayEPA MRID Number 48827601Page47of80 DER Template Version: 22 September 2011Abbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weightedby the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.3Hind-limb length is normalized to snout-vent length (SVL).Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Table 17: Growth Endpoints 1,2 in the AMA with EPTC.

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, effects are reported based on comparison to the negative (clean water) control.

2 Unless otherwise specified, effects are considered statistically significant at p < 0.05.

E. Study Deficiencies

There were no major deficiencies noted in this study. Although there were minor deviations from guidelines, as noted in the Materials and Methods section of the DER, the study met all validity and
performance criteria.

F. Reviewer’s Comments

The reviewer’s results were based on replicate mean s and medians calculated by excluding tadpoles at
NF stage  61. Therefore, the reviewer’s results are reported in the Executive Summary and Conclusions

sections of this DER.

The in-life portion of the definitive toxicity test was conducted from February 14 to March 6, 2012. Treatment
(mg a.i./L)

[measured] Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

% Diff. p % Diff. p %

Diff. p %

Diff. p

Negative Control 0 NA 0 NA 0 NA 0 NA

0.019 -5.8 0.07 -2.2 0.04 -12.2 0.12 -1.5 0.19

0.10 -6.7 0.02 -3.2 0.01 -16.1 0.03 -5.9 0.02

0.50 -7.8 0.006 -5.1 <0.001 -22.2 0.006 -12.0 <0.001

2.6 -15.2 <0.001 -8.5 <0.001 -41.9 <0.001 -22.9 <0.001

Statistical Test Jonckheere Jonckheere Jonckheere JonchkeereData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 III. REFERENCES

APHA, AWWA, WPCF, 2005. Standard Methods for the Examination of Water and Wastewater. 21st Edition, Washington, DC.

ASTM, 2004. Standard Guide for Conducting the Frog Embryo Teratogenesis Assay – Xenopus (FETAX).
American Society for Testing and Material s, ASTM E 1439-98(2004). Philadelphia, PA.

ASTM, 2005. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM Desi gnation E: 1241-05. American Society for Testing and Materials, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, Pennsylvania.

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information System TM . User’s Guide.
Tidepool Scientific Software, McKinleyville, California.

Mount, D.I. and W.A. Brungs, 1967. A simplified dosing apparatus for fish toxicological studies. Water

Research 1L 21-29.

Nieuwkoop, P.D. and Faber, J. 1994. Normal Table of Xenopus laevis. Garland Publishing, New York.

OECD, 1998. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring. Number 1. OECD Principles on Good Laboratory Practice (as revised in 1997). Environment Directorate Chemicals Group and Management Committee. ENV/MC/CHEM(98)17. OECD Paris. France. 41 pp.

OECD, 2006. Current Approaches in t he Statistical Analysis of Ecotoxicit y Data: A Guidance to Application. Environmental Health and Safety Publications. Series on Testing and Assessment. No. 54. Paris, France.

OECD, 2009. Amphibian Metamorphosis Assay. OECD Gu ideline for the Testing of Chemicals. No. 231.
Paris, France.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 50 of 80

DER Template Version: 22 September 2011 Sprague, J.B. 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity. Water

Research 3: 793-821.

U.S. EPA, 1989. 40 CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good Laboratory Practices Standards; Final Rule. Office of the Federal Register, National Archives and Records

Administration. U.S. Government Pr inting Office, Washington, D.C.

U.S. EPA, 1989. Toxic Substances Control Act; Good Laboratory Practi ce Standards; Final Rule. (40 CFR, Part 792) Federal Register, Part III, 48(230): 53922-53944, August 17, 1989.

U.S. EPA, 1996. Office of Prevention, Pesticides and Toxic Substances. Ecological Effects Guideline, OPPTS 850.1000. Special Consideration for Conducting Aquatic Laboratory Studies. "Public Draft". EPA
712-C-96-113. April 1996. U. S. Environmental Protection Agency, Washington, D.C.

U.S. EPA, 2007. Office of Waste. Test Methods for Evaluating Solid Waste, Physical/Chemical Methods (SW-846). February 2007. U.S. Environmental Protection Agency, Washington, D.C.

U.S. EPA, 2009. Endocrine Disrupter Screening Program Test Guidelines OPPTS 890.1100: Amphibian Metamorphosis (Frog). EPA 740-C-09-002. October 2009.

York, Duncan, 2012. EPTC - Short-Term Reproduction Assay with Fathead Minnow (Pimephales promelas)
Following OPPTS 890.1350 and OECD 229 Guidelines. Smithers Viscient, Wareham,MA. Study No.

12791.6156.Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 

### APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR01 ( 7-d wet weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.958 0.504 0.999 0.439 USE PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.57 0.07 0.04 12.33 0.46, 0.69 Dose1 4 0.50 0.07 0.04 14.76 0.39, 0.62 Dose2 4 0.48 0.04 0.02 7.87 0.42, 0.54 Dose3 4 0.45 0.04 0.02 9.56 0.38, 0.51 Dose4 4 0.33 0.06 0.03 16.77 0.24, 0.42

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.57 0.50 0.67 . . Dose1 0.50 0.42 0.59 87.80 12.20 Dose2 0.48 0.44 0.52 83.93 16.07 Dose3 0.46 0.39 0.49 77.79 22.21 Dose4 0.31 0.29 0.42 58.10 41.90

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 9.23 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.57 . 0.57 . . . . . . Dose1 0.50 0.299 0.50 0.064 . . . . . Dose2 0.48 0.122 0.48 0.024 0.981 . . . . Dose3 0.45 0.024 0.45 0.004 0.638 0.908 . . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 Dose4 0.33 <.001 0.33 <.001 0.007 0.019 0.093 . .

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 13.03 0.011

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.57 . . Dose1 0.50 0.346 0.124 Dose2 0.48 0.103 0.029 Dose3 0.46 0.067 0.006 Dose4 0.31 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 9.23 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.57 . -0.47 . . . . . . Dose1 -0.50 0.299 -0.47 0.997 . . . . . Dose2 -0.48 0.122 -0.47 0.998 0.981 . . . . Dose3 -0.45 0.024 -0.47 0.999 0.638 0.908 . . . Dose4 -0.33 <.001 -0.47 0.999 0.007 0.019 0.093 . .

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 13.03 0.011Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.57 . . Dose1 -0.50 0.346 0.876 Dose2 -0.48 0.103 0.971 Dose3 -0.46 0.067 0.994 Dose4 -0.31 0.067 1.000

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR02 ( 7-d stage (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.811 0.001 6.750 0.003 USE NON-PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 54.00 0.00 0.00 0.00 . , . Dose1 4 53.75 0.50 0.25 0.93 52.95, 54.55 Dose2 4 54.00 0.00 0.00 0.00 . , . Dose3 4 54.00 0.00 0.00 0.00 . , . Dose4 4 53.25 0.50 0.25 0.94 52.45, 54.05

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 54.00 54.00 54.00 . . Dose1 54.00 53.00 54.00 99.54 0.46 Dose2 54.00 54.00 54.00 100.00 0.00 Dose3 54.00 54.00 54.00 100.00 0.00 Dose4 53.00 53.00 54.00 98.61 1.39

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 4.25 0.017

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 54.00 . 54.00 . . . . . . Dose1 53.75 0.638 53.92 0.424 . . . . . Dose2 54.00 1.000 53.92 0.453 0.795 . . . . Dose3 54.00 1.000 53.92 0.468 0.795 1.000 . . . Dose4 53.25 0.015 53.25 0.003 0.219 0.030 0.030 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 10.09 0.039

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 54.00 . . Dose1 54.00 0.478 0.159 Dose2 54.00 1.000 0.500 Dose3 54.00 1.000 0.673 Dose4 53.00 0.102 0.027

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4 Jonckheere Dose4

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 4.25 0.017

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -54.00 . -53.80 . . . . . . Dose1 -53.75 0.638 -53.80 0.879 . . . . . Dose2 -54.00 1.000 -53.80 0.903 0.795 . . . . Dose3 -54.00 1.000 -53.80 0.915 0.795 1.000 . . . Dose4 -53.25 0.015 -53.80 0.922 0.219 0.030 0.030 . .

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 10.09 0.039

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -54.00 . . Dose1 -54.00 0.478 0.841 Dose2 -54.00 1.000 0.500 Dose3 -54.00 1.000 0.327 Dose4 -53.00 0.102 0.973

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR03 ( 7-d sn-vent length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.962 0.589 0.936 0.470 USE PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 20.13 0.19 0.09 0.94 19.82, 20.43 Dose1 4 18.95 1.03 0.51 5.42 17.31, 20.59 Dose2 4 18.78 0.67 0.33 3.54 17.72, 19.83 Dose3 4 18.55 0.66 0.33 3.54 17.51, 19.59 Dose4 4 17.08 0.78 0.39 4.57 15.83, 18.32

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 20.05 20.00 20.40 . . Dose1 18.95 17.70 20.20 94.16 5.84 Dose2 18.60 18.20 19.70 93.29 6.71 Dose3 18.45 17.90 19.40 92.17 7.83 Dose4 17.10 16.10 18.00 84.84 15.16

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 9.25 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 20.13 . 20.13 . . . . . . Dose1 18.95 0.107 18.95 0.020 . . . . . Dose2 18.78 0.057 18.78 0.010 0.997 . . . . Dose3 18.55 0.024 18.55 0.004 0.930 0.991 . . . Dose4 17.08 <.001 17.08 <.001 0.016 0.030 0.070 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 12.90 0.012

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 20.05 . . Dose1 18.95 0.233 0.073 Dose2 18.60 0.066 0.017 Dose3 18.45 0.066 0.006 Dose4 17.10 0.066 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose2

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 9.25 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -20.13 . -18.70 . . . . . . Dose1 -18.95 0.107 -18.70 0.998 . . . . . Dose2 -18.78 0.057 -18.70 0.999 0.997 . . . . Dose3 -18.55 0.024 -18.70 0.999 0.930 0.991 . . . Dose4 -17.08 <.001 -18.70 0.999 0.016 0.030 0.070 . .

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 12.90 0.012

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -20.05 . . Dose1 -18.95 0.233 0.927 Dose2 -18.60 0.066 0.983 Dose3 -18.45 0.066 0.994 Dose4 -17.10 0.066 1.000

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR04 ( 7-d hind-limb length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.951 0.379 1.332 0.304 USE PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 2.31 0.08 0.04 3.37 2.18, 2.43 Dose1 4 2.07 0.21 0.11 10.29 1.73, 2.41 Dose2 4 2.05 0.18 0.09 8.98 1.75, 2.34 Dose3 4 1.99 0.11 0.05 5.33 1.82, 2.16 Dose4 4 1.81 0.10 0.05 5.75 1.64, 1.98

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 2.28 2.26 2.42 . . Dose1 2.10 1.80 2.30 89.80 10.20 Dose2 2.10 1.78 2.20 88.63 11.37 Dose3 2.00 1.88 2.08 86.20 13.80 Dose4 1.81 1.69 1.93 78.42 21.58

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 5.97 0.004

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 2.31 . 2.31 . . . . . . Dose1 2.07 0.116 2.07 0.022 . . . . . Dose2 2.05 0.072 2.05 0.014 0.999 . . . . Dose3 1.99 0.026 1.99 0.005 0.926 0.981 . . . Dose4 1.81 <.001 1.81 <.001 0.135 0.207 0.445 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 11.23 0.024

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 2.28 . . Dose1 2.10 0.235 0.074 Dose2 2.10 0.067 0.020 Dose3 2.00 0.067 0.004 Dose4 1.81 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose2

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 5.97 0.004

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -2.31 . -2.05 . . . . . . Dose1 -2.07 0.116 -2.05 0.996 . . . . . Dose2 -2.05 0.072 -2.05 0.998 0.999 . . . . Dose3 -1.99 0.026 -2.05 0.998 0.926 0.981 . . . Dose4 -1.81 <.001 -2.05 0.999 0.135 0.207 0.445 . .

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 11.23 0.024

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -2.28 . . Dose1 -2.10 0.235 0.926 Dose2 -2.10 0.067 0.980 Dose3 -2.00 0.067 0.996 Dose4 -1.81 0.067 1.000

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR05 ( 7-d norm hind-limb )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.935 0.191 5.166 0.008 USE NON-PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.12 0.00 0.00 2.28 0.11, 0.12 Dose1 4 0.11 0.01 0.00 5.08 0.10, 0.12 Dose2 4 0.11 0.01 0.00 8.53 0.09, 0.12 Dose3 4 0.11 0.00 0.00 2.21 0.10, 0.11 Dose4 4 0.11 0.00 0.00 1.64 0.10, 0.11

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.11 0.11 0.12 . . Dose1 0.11 0.10 0.11 94.14 5.86 Dose2 0.11 0.10 0.12 94.79 5.21 Dose3 0.11 0.11 0.11 92.62 7.38 Dose4 0.11 0.10 0.11 91.54 8.46

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 2.13 0.127

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.12 . 0.12 . . . . . . Dose1 0.11 0.236 0.11 0.059 . . . . . Dose2 0.11 0.325 0.11 0.062 1.000 . . . . Dose3 0.11 0.104 0.11 0.021 0.988 0.956 . . . Dose4 0.11 0.055 0.11 0.010 0.919 0.838 0.997 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

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DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 7.19 0.126

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.11 . . Dose1 0.11 0.125 0.030 Dose2 0.11 0.678 0.170 Dose3 0.11 0.066 0.038 Dose4 0.11 0.065 0.010

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 2.13 0.127

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.12 . -0.11 . . . . . . Dose1 -0.11 0.236 -0.11 0.975 . . . . . Dose2 -0.11 0.325 -0.11 0.983 1.000 . . . . Dose3 -0.11 0.104 -0.11 0.987 0.988 0.956 . . . Dose4 -0.11 0.055 -0.11 0.988 0.919 0.838 0.997 . .

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 7.19 0.126

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 65 of 80

DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.11 . . Dose1 -0.11 0.125 0.970 Dose2 -0.11 0.678 0.830 Dose3 -0.11 0.066 0.962 Dose4 -0.11 0.065 0.990

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 66 of 80

DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR06 ( 21-d stage (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.822 0.002 19.000 <.001 USE NON-PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 57.00 0.00 0.00 0.00 . , . Dose1 4 57.25 0.50 0.25 0.87 56.45, 58.05 Dose2 4 57.00 0.00 0.00 0.00 . , . Dose3 4 57.50 0.58 0.29 1.00 56.58, 58.42 Dose4 4 57.00 0.00 0.00 0.00 . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 57.00 57.00 57.00 . . Dose1 57.00 57.00 58.00 100.44 -0.44 Dose2 57.00 57.00 57.00 100.00 0.00 Dose3 57.50 57.00 58.00 100.88 -0.88 Dose4 57.00 57.00 57.00 100.00 0.00

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 1.71 0.199

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 57.00 . 57.19 . . . . . . Dose1 57.25 0.693 57.19 0.852 . . . . . Dose2 57.00 1.000 57.19 0.879 0.835 . . . . Dose3 57.50 0.165 57.19 0.892 0.835 0.282 . . . Dose4 57.00 1.000 57.00 0.648 0.835 1.000 0.282 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 67 of 80

DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 5.96 0.202

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 57.00 . . Dose1 57.00 0.478 0.841 Dose2 57.00 1.000 0.500 Dose3 57.50 0.223 0.917 Dose4 57.00 1.000 0.667

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 1.71 0.199

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -57.00 . -57.00 . . . . . . Dose1 -57.25 0.693 -57.13 0.365 . . . . . Dose2 -57.00 1.000 -57.13 0.390 0.835 . . . . Dose3 -57.50 0.165 -57.25 0.209 0.835 0.282 . . . Dose4 -57.00 1.000 -57.25 0.213 0.835 1.000 0.282 . .

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 5.96 0.202

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 68 of 80

DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -57.00 . . Dose1 -57.00 0.478 0.159 Dose2 -57.00 1.000 0.500 Dose3 -57.50 0.223 0.083 Dose4 -57.00 1.000 0.333

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 69 of 80

DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.959 0.530 1.218 0.344 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.92 0.11 0.06 5.77 1.74, 2.09 Dose1 4 1.89 0.02 0.01 1.22 1.85, 1.92 Dose2 4 1.80 0.07 0.03 3.74 1.70, 1.91 Dose3 4 1.69 0.08 0.04 4.99 1.55, 1.82 Dose4 4 1.48 0.05 0.03 3.48 1.39, 1.56

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.93 1.77 2.03 . . Dose1 1.89 1.86 1.91 98.51 1.49 Dose2 1.80 1.73 1.89 94.11 5.89 Dose3 1.69 1.58 1.78 88.02 11.98 Dose4 1.46 1.44 1.55 77.06 22.94

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 23.69 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.92 . 1.92 . . . . . . Dose1 1.89 0.949 1.89 0.353 . . . . . Dose2 1.80 0.139 1.80 0.028 0.507 . . . . Dose3 1.69 0.002 1.69 <.001 0.011 0.216 . . . Dose4 1.48 <.001 1.48 <.001 <.001 <.001 0.008 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 70 of 80

DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 15.19 0.004

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 1.93 . . Dose1 1.89 0.494 0.193 Dose2 1.80 0.235 0.020 Dose3 1.69 0.103 <.001 Dose4 1.46 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 23.69 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.92 . -1.75 . . . . . . Dose1 -1.89 0.949 -1.75 0.999 . . . . . Dose2 -1.80 0.139 -1.75 1.000 0.507 . . . . Dose3 -1.69 0.002 -1.75 1.000 0.011 0.216 . . . Dose4 -1.48 <.001 -1.75 1.000 <.001 <.001 0.008 . .

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 15.19 0.004

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 71 of 80

DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -1.93 . . Dose1 -1.89 0.494 0.807 Dose2 -1.80 0.235 0.980 Dose3 -1.69 0.103 1.000 Dose4 -1.46 0.067 1.000

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 72 of 80

DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.943 0.277 3.367 0.037 USE NON-PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 31.15 0.68 0.34 2.17 30.07, 32.22 Dose1 4 30.45 0.24 0.12 0.79 30.06, 30.83 Dose2 4 30.15 0.52 0.26 1.71 29.33, 30.97 Dose3 4 29.56 0.41 0.21 1.39 28.91, 30.21 Dose4 4 28.50 0.35 0.17 1.22 27.95, 29.06

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 31.17 30.50 31.74 . . Dose1 30.37 30.26 30.80 97.76 2.24 Dose2 30.07 29.67 30.78 96.80 3.20 Dose3 29.62 29.01 30.00 94.91 5.09 Dose4 28.39 28.22 29.00 91.52 8.48

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 18.49 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 31.15 . 31.15 . . . . . . Dose1 30.45 0.149 30.45 0.029 . . . . . Dose2 30.15 0.027 30.15 0.005 0.886 . . . . Dose3 29.56 <.001 29.56 <.001 0.099 0.410 . . . Dose4 28.50 <.001 28.50 <.001 <.001 0.001 0.038 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 73 of 80

DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 15.70 0.003

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 31.17 . . Dose1 30.37 0.156 0.042 Dose2 30.07 0.156 0.014 Dose3 29.62 0.067 <.001 Dose4 28.39 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose1

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 18.49 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -31.15 . -29.96 . . . . . . Dose1 -30.45 0.149 -29.96 1.000 . . . . . Dose2 -30.15 0.027 -29.96 1.000 0.886 . . . . Dose3 -29.56 <.001 -29.96 1.000 0.099 0.410 . . . Dose4 -28.50 <.001 -29.96 1.000 <.001 0.001 0.038 . .

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 15.70 0.003

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 74 of 80

DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -31.17 . . Dose1 -30.37 0.156 0.958 Dose2 -30.07 0.156 0.986 Dose3 -29.62 0.067 1.000 Dose4 -28.39 0.067 1.000

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 75 of 80

DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.975 0.850 0.721 0.591 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 13.64 0.37 0.18 2.69 13.05, 14.22 Dose1 4 13.74 0.71 0.36 5.19 12.61, 14.87 Dose2 4 13.30 0.54 0.27 4.08 12.44, 14.16 Dose3 4 12.90 0.97 0.48 7.52 11.36, 14.45 Dose4 4 10.39 0.44 0.22 4.22 9.69, 11.09

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 13.77 13.10 13.91 . . Dose1 13.63 12.99 14.71 100.74 -0.74 Dose2 13.15 12.82 14.07 97.51 2.49 Dose3 13.05 11.61 13.91 94.61 5.39 Dose4 10.44 9.83 10.85 76.16 23.84

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 18.51 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 13.64 . 13.69 . . . . . . Dose1 13.74 0.998 13.69 0.629 . . . . . Dose2 13.30 0.867 13.30 0.299 0.866 . . . . Dose3 12.90 0.340 12.90 0.081 0.390 0.904 . . . Dose4 10.39 <.001 10.39 <.001 <.001 <.001 <.001 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 76 of 80

DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 10.41 0.034

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 13.77 . . Dose1 13.63 0.678 0.282 Dose2 13.15 0.494 0.121 Dose3 13.05 0.494 0.068 Dose4 10.44 0.067 <.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4 Jonckheere Dose4

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 18.51 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -13.64 . -12.79 . . . . . . Dose1 -13.74 0.998 -12.79 0.982 . . . . . Dose2 -13.30 0.867 -12.79 0.988 0.866 . . . . Dose3 -12.90 0.340 -12.79 0.991 0.390 0.904 . . . Dose4 -10.39 <.001 -12.79 0.992 <.001 <.001 <.001 . .

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 10.41 0.034

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 77 of 80

DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -13.77 . . Dose1 -13.63 0.678 0.718 Dose2 -13.15 0.494 0.879 Dose3 -13.05 0.494 0.932 Dose4 -10.44 0.067 0.999

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 78 of 80

DER Template Version: 22 September 2011 test for amphib metamorph screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.964 0.626 0.632 0.647 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.44 0.02 0.01 3.42 0.42, 0.46 Dose1 4 0.45 0.03 0.01 5.86 0.41, 0.49 Dose2 4 0.44 0.03 0.01 5.85 0.40, 0.48 Dose3 4 0.44 0.04 0.02 8.23 0.38, 0.50 Dose4 4 0.36 0.02 0.01 4.40 0.34, 0.39

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.43 0.43 0.46 . . Dose1 0.45 0.42 0.49 102.50 -2.50 Dose2 0.44 0.42 0.48 100.23 -0.23 Dose3 0.44 0.39 0.48 99.77 0.23 Dose4 0.36 0.34 0.38 82.49 17.51

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 8.23 0.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.44 . 0.45 . . . . . . Dose1 0.45 0.924 0.45 0.708 . . . . . Dose2 0.44 1.000 0.44 0.642 0.978 . . . . Dose3 0.44 1.000 0.44 0.612 0.959 1.000 . . . Dose4 0.36 0.002 0.36 <.001 0.001 0.004 0.005 . .Data Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 79 of 80

DER Template Version: 22 September 2011 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 9.59 0.048

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.43 . . Dose1 0.45 0.678 0.718 Dose2 0.44 0.889 0.442 Dose3 0.44 0.678 0.500 Dose4 0.36 0.066 0.017

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose4 Jonckheere Dose4

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 4 15 8.23 0.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.44 . -0.43 . . . . . . Dose1 -0.45 0.924 -0.43 0.844 . . . . . Dose2 -0.44 1.000 -0.43 0.872 0.978 . . . . Dose3 -0.44 1.000 -0.43 0.886 0.959 1.000 . . . Dose4 -0.36 0.002 -0.43 0.894 0.001 0.004 0.005 . .

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 4 9.59 0.048

MannWhit - testing each trt median signif. different from controlData Evaluation Record on the Toxicity of EPTC to Amphibians, Metamorphosis Assay

EPA MRID Number 48827601

Page 80 of 80

DER Template Version: 22 September 2011 Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.43 . . Dose1 -0.45 0.678 0.282 Dose2 -0.44 0.889 0.558 Dose3 -0.44 0.678 0.500 Dose4 -0.36 0.066 0.983

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1150, Androgen Recep tor Binding (Rat Prostate Cytosol)

EPA Contract No. EP10H001452 Task Assignment No. 2-13-2012 (MRID 48616403)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: David A. McEwen, B.S. Date: 1/23/2012
Secondary Reviewer Signature: Michelle Sharpe-Kass, M.S Date: 1/24/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 1/27/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 1/27/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).EPTC I 041401 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page I of 16 OCSPP 890.1150/ OECD None


### Primary Reviewer: Jessica Ryman, Ph.D., D.A.B.T 

### Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D. 

### Health Effects Division Sig """" : --J-+-- .P......,..-.;..-

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### Date:**-~--'~~c..L..,J..-­** 

### Signature:**-~""t""'~,L-----,----­** 

### Date:

__**___,,,6,,,_._. lt** **~}J~** Template version 08/201 t



### DATA EVALUATION RECORD

STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol); OCSPP 890.1150



### PC CODE: 041401 

### DP BARCODE: D397177



### TXR#: 0051762 

### CAS No.: 759-94-4



### TEST MATERIAL (PURITY): EPTC ( > 99.9% a.i.)



### SYNONYMS: S-ethyl dipropylthiocarbamate



### CITATION: Willoughby, J.A (2011). EPTC: Androgen Receptor Binding (Rat Prostate Cytosol). CeeTox, Inc
., Kalamazoo
, MI. Laboratory Study # : 9142V100349ARB, November 10
, 2011. MRID 48616403. Unpublished.

Willoughby, J.A. (2012). Supplemental Information

- Laboratory Proficiency Data for ERTA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc.
, Kalamazoo
, MI. Julyl 9
, 2012. MRID 48843501. Unpublished.



### SPONSOR: Gowan Company, 370 S. Main Street, Yuma, AZ



### TEST ORDER#: EDSP-041401-64



### EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48616403)
, ve ntral prostate cytosol from Sprague Dawley rats was used as the source of the AR. The saturation binding experiment (data submitted separately, MRID 48843501) was conducted
to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radio labeled reference androgen (Rl 881 ).

Saturation binding data were not originally provided in the study report; however
, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The mean dissociation constant (Kct) for [3H]-R1881 was 1.080 ± 0.690 nM
, and the estimated Bmax was 1.071±0.167 fmo l/100 μg protein for the single batch of prostate cytosol that was used in the assay. Only one of the three Kct values was within the range reported in the EPA validation program (0.685 to 1.57 nM)
, and all Bmax values were below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for individual runs acceptable ( R 2 

## = 0.880-0.986)
, confidence in these numbers is low based on the large variation in Kct estimates among runs

.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None


### The competitive binding assay was conducted to measure the binding of a single concentration (1 nM) of the radio-labeled reference androgen ([ 3 

### H]-R1881) in the pres ence of increasing concentrations of EPTC (l ogarithmic increase from 10 -10 

### to 10 -3 

### M). DMSO was used as a vehicle at a final assay concentration of approximately 3.2%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand reference standard. Three independent runs were conducted with 3 replicates per concentration per run.

The estimated log IC 50 

### s for R1881 and dexamethasone were with in the excepted ranges at -9.0 to -9.9 M and -4.4 to -4.6 M, resp ectively. The average log IC 50 

### was -9.3 M for R1881 and -4.5 M for dexamethasone, and the mean RBA for the weak positive control was 0.0016%. Confidence in the numbers is high due to the small variati on, and the solvent contro l responses indicated no drift in the study assay. The perf ormance criteria were met, with the following exceptions: in the second assay, the top specific binding (%) of EPTC was 118.2%, which slightly exceeded the range of the Guideline performance criteria ( 80 to 115%); and in the first assay, the bottom plateau level (% binding) for dexamethasone was not able to be accurately calculated, because the concentration range for dexamethasone was prepar ed slightly different than the guideline, in error. As these deviations were minor and were not considered to have affected the results of the study, all three runs we re considered valid.

EPTC reduced the mean specific binding of the ligand in a concentration dependent manner in all three runs, with maximum disp lacement of binding occurring at 10 -3 

### M (50-56.5% binding). An IC 50 

### and relative binding affinity (RBA) could not be calculated for EPTC. Specific binding was also reduced at 10 -4 

### M (87.4-91%), but not at EPTC concentrations ≤ 10 -5 

### M ( ≥ 92.8% binding). Specific [ 3 

### H]-R1881 binding to the AR was between 50 and 75% at all concentrations of EPTC tested in all three runs.

Based on the results from the three runs, EPTC is classified as Equivocal in the Androgen Receptor Binding Assay.

The assay satisfies the EDSP Tier 1 Test Order require ments for an Androgen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance and Quality Assurance statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test facility: CeeTox, Inc.
Location: 4717 Campus Drive, Kalamazoo, MI 49008 Study Director: J.A. Willoughby, Sr. Other Personnel: D. Blakeman, C. Haines, and S. McColley Study Period: May 25, 2011 to November 10, 2011



### 2. Test substance: EPTC
Description: Liquid Source: Gowan company Batch/Lot #: P1105096 Purity: >99.9% (expiry date February 4, 2015) Solubility: Not reported Volatility: Not reported Stability: Not reported Storage conditions: Ambient CAS #: 759-94-4 Molecular weight: 189.3 g/mol Structure:

N S O CH

3

CH 3 CH 3



### 3. Non-labeled ligand: R1881 Supplier: Sigma Aldrich, St. Louis, MO Catalog #: R0908 Batch #: 060M4638 Purity: 98% CAS #: 965-93-5


### 4. Radioactive ligand: [ 3 

### H]-R1881 Supplier: Perkin-Elmer, Boston, MA Catalog and Batch #: NET590; Lot # 653698 Date of production: February 24, 2011 Date of use: September 10, 2011 to September 17, 2011 Radiochemical purity: Not reported Specific activity: 85.1 Ci/mol **The SA adjusted was 82.6 Ci/mol (10-Sept-2011), 82. 5 Ci/mol (13-Sept-2011), and 92.5 Ci/mol (17-Sept-2011). Concentration in stock: 1.0 nM



### 5. Positive control: Dexamethasone Supplier: Sigma Aldrich, St. Louis, MO Catalog #: D1756 Lot #: 077K1050 Purity: 99% CAS # : 50-02-2Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None



### 6. Solvent/vehicle control: DMSO
Justification for choice of solvent: Due to issues with pipetting and volatilization when using ethanol. Final concentration: 3.2%



### B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was purchased from Charles River La boratories (location not report ed). Male Sprague Dawley rats (number not reported) were castrated at 90 days of age and were euthanized the following day. The ventral prostate tissues we re collected and stored at approximately - 80°C until use, and were processed as a batch and used for multiple studies.

The cytosol was prepared by adding low-salt TEDG buffer (10 mM Tris, 1 mM sodium molybdate, 1.5 mM EDTA, 10% gly cerol and 1 mM DTT, 0.5% pr otease inhibitor) at pH 7.4 to the ventral prostate ti ssues at 10 mL/g of tissue. The tissues were minced, homogenized at 4°C, and cen trifuged for 30 min at 30,000 × g at 4°C. The supernatant was aliquoted (2-4 mL) in tubes and stored at - 80°C until used. Protein concentration of the cytosol was determined to be 6.2 mg/mL using a protein kit compatible with DTT in the TEDG buffer (BioRad Protein Assay Kit).

2. Saturation Radioligand Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [ 3 

### H]-R1881 was performed to demonstrate that the AR was present in reasonabl e concentrations and had the appropriate affinity for the R1881 ligand (MRID 48843501). The conditions for the saturation binding experiment are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a Source of receptor Rat ventral prostate cytosol Concentration of radioligand (as serial dilutions) 0.25 to 10 nM Concentration of non-labeled ligand (100X [radioligand]) 2 to 1000 nM Optimization of receptor concentration Sufficient to bind 25 to 35% of radioligand at 0.25 nM Temperature 4o C Incubation time 16 to 20 hours Composition of assay buffer (TEDG) Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride (PMSF) 1 mM DTT 1 mM Sodium Molybdate 1 mM a Data were obtained from page 2 of the study report (MRID 48843501).



### On the day of each assay, the specif ic activity of the stock solution [ 3 

### H]-R1881 (originally 85.1 Ci/mmol as manufactured on February 24, 2011) was adjusted for decay over time (adjusted specific activities were not reported), and seri al dilutions in low-salt TEDG+PMSF buffer were prepared to achieve th e final concentrations in cytosol of 0.25,0.50, 0.70, 1.0, 1.5, 2.5, 5.0, and 10 nM to determine total binding. To determine nonAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None


### specific binding, solutions of non-labeled R1881 were prepared in a similar manner to achieve concentrations that were 100-fold greater than each re spective radiolabeled concentration, resulting in final concentr ations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, th e radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

### H]-R1881 and 7.5, 15, or 30 μL of 100 nM [ 3 

### H]-R1881 was measured.

For each batch of cytosol, the optimal protei n concentration was determined by calculating specific binding to differing amounts of protei n per tube with 0.25 nM radiolabeled R1881. The optimal protein concentration was determin ed to be 1.86 mg protein/assay tube, which resulted in the binding of approximately 25-35% of the total radioactivity added. Cytosolic protein used in this assay was thawed fresh fo r this experiment at 4°C, and maintained at 4°C during the binding assay. Each saturation binding experiment consisted of three noncurrent runs (conducted on September 9, 11, and 16, 2011, respectively), and each run contained three replicates at each concentration, re sulting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a,b Total Binding Non-Specific Binding Radioligand alone Tubes 1-24 c Tubes 25-48 d Tubes 49-72 e [ 3 H]-R1881 Final conc. (nM) [ 3 H]-R1881 Final conc. (nM) R1881 Final conc. (nM) [ 3 H]-R1881 Initial conc. (nM) [ 3 H]-R1881 (μL) 0.25 0.25 25 10 7.5 0.50 0.50 50 10 15 0.70 0.70 70 10 21 1.0 1.0 100 10 30 1.5 1.5 150 10 45 2.5 2.5 250 100 7.5 5.0 5.0 500 100 15 10 10 1000 100 30 a Data were obtained from page 3 of the study report (MRID 48843501). b Each concentration was run in triplicate for a total of 72 samples. c Tubes 1-24 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 H]-R1881. Samples were dried, and 300 μl of prostate cytosol were added. d Tubes 25-48 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 H]-R1881. R1881 was added in a 100-fold mola r excess of [ 3 H]-R1881 in a volume of 7.5-45 μL. Samples were dried, and 300 μl of prostate cytosol were added. e Tubes 49-72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 H]-R1881 or 7.5, 15, 21, or 30 μL of 100 nM [ 3 H]-R1881 without cytosol or other component s to determine the total counts added.



### Following addition of triamcinolone acetonide, [ 3 

### H]-R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 20 hours at approximately 4°C. Samples were maintained at temperatures of approximately 4°C except during whole rack vortexing. To separa te bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed 5 times with 4-minute intervals over 20 minutes. The samples were then centrifuged, and the s upernatant was aspirate d and discarded. The samples were washed 4 times in 2 mL of ice cold TEDG+PMSF buffer, followed by vortexing and centrifugation for 3 minutes at 700 x g. Following the last wash and decanting of the Tris buffer pellets were then extracted by additional of 2 mL of ethanol. The samples were vortexed and centrifuged for 10 minutes at 1000 x g. Samples wereAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None


### maintained on ice at all times between vortexing . After the final centrifugation, the ethanol supernatants were decanted into scintillation vials that each contained 14-mL portions of scintillation cocktail, and the radiation was quantified by liquid scin tillation counting. A total of three runs were performed.

3. Competitive Binding Experiment: A summary of the assay conditions for the competitive binding experiment is presented in Table 3.

TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat prostate cytosol Concentration of radioligand 1 nM Optimization of receptor concentr ation Sufficient to bind 10-15% b Concentration of test substance (as serial dilutions) 10 -10 to 10 -3 M Incubation Temperature 4±2°C Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% (v/v) Protease inhibitor 0.5% (v/v) DTT 1 mM Sodium molybdate 1 mM a Data were obtained from page 15 of the study report. b Stated in Study Report on page 15.



### The competitive binding experiment was performed according to the protocol provided in the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures the binding of a singl e concentration of [ 3 

### H]-R1881 (adjusted specific activity of 85.1 Ci/mol) to the AR in the presence of incr easing concentrations of a test substance.

DMSO was used as the vehicle, and the solu bility of the test material at the top concentration of 10 -3 

### M in 100% DMSO was determined by visual observation. Additionally, all test material solutions were examined for precipitation following the overnight incubation period. As no precip itation was observed, the suitable top concentration of EPTC for use in all independent runs was 10 -3 

### M.

Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (DMSO) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days, and each run contained three replicates at e ach concentration, resul ting in a total of 81 samples per run.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None

TABLE 4. Competitor Final Molar (M) Con centrations in Competitive Binding Assay a, b

Solvent Control Re ference standard c Weak positive control d Test Chemical None DMSO R1881 Dexamethasone EPTC Tubes 7-12 Tubes 13-18 and 19-33 e Tubes 37-60 Tubes 61-84 Tubes 1-6 1×10 -12.48 1×10 -11 1×10 -11 1×10 -10 1×10 -10 1×10 -11.48 1×10 -10 1×10 -10 1×10 -9 1×10 -9 1×10 -10.48 1×10 -9 1×10 -9 1×10 -8 1×10 -8
1×10 -9.48 1×10 -8 1×10 -8 1×10 -7 1×10 -7
1×10 -8.48 1×10 -7 1×10 -7 1×10 -6 1×10 -6
1×10 -7.48 1×10 -6 1×10 -6 1×10 -5 1×10 -5
-- 1×10 -5 1×10 -4 1×10 -4
-- 1×10 -4 1×10 -3 1×10 -3 a Data were obtained from pages 34-39 of the study report. b Each concentration of each chemical was run in triplicat e for a total of 81 tubes per run. Tubes 7-84 contained

50 μL of triamcinolone acetonide and 30 μL [ 3 H]-R1881. Samples were dried, and 300 μL of prostatecytosol were added. Tubes 7-84 also contained 10 μL of the solvent control, reference standard (nonradiolabeled R-1881), weak positive control, or test substance, with the exception of Tubes 13-18 that contained 30 μL of non-radiolabeled R1881 (used to evaluate non-specific binding). Tubes 1-6 contained only 30 μL of [ 3 H]-R1881. c The concentrations of R1881 used in the 1 st competitive binding assay ranged from 1x10 -7.48 to 1x10 -12.48

, while the standard concentrations of 1x10 -6 to 1x10 -11 were used in the 2 nd and 3 rd assays. d The concentrations of dexamethasone used in the 1 st competitive binding assay ranged from 1x10 -4 to 1x10 -11

, while concentrations of 1x10 -3 to 1x10 -10 were used in the 2 nd and 3 rd assays. e Tubes 13-18 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881



### 4. Data Analysis: The top and bottom of the curve, H ill slope, inhibition concentration (IC 50 

### ), and standard deviations were assessed using GraphPad PRISM (GraphPad Software, Inc., San Diego, CA).

5. Definitions

a. Classification of test material

If the data fit a 4-parameter non linear regression model, the test chemical is classified as:

Binder: The average curve for the test chemical across runs crosses 50% of radioligand bound ( i.e. a Log IC 50 

### was obtained).

Non-Binder: The average lowest portion of curves acr oss runs is greate r than 75% activity ( i.e. less than 25% displacement of radioliga nd), or the data do not fit the model.

Equivocal: The average lowest portion of curves across runs is between 50% and 75% radioligand binding ( i.e. radioligand displacement is at least 25% but less than 50%), or the curve falls outside the range for the weak positive control (-0.6 to -1.4).

b. Descriptors for receptor binding

B max 

### : Maximal binding capacity K d 

### : Dissociation constantsAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None



### IC 50 

### : Concentration of the test substance at which 50% of radioligand is displaced from the AR by the competitor 

### Relative Binding Affinity (RBA): IC 50 

### of R1881 × 100 ÷ IC 50 

### of test substance

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5. The mean K d 

### for [ 3 

### H]-R1881 was 1.080±0.690 nM, and the estimated B max 

### was 1.071±0.167 fmol/100 μg protein for th e single batch of prostate cytosol that was used in the assay. Only one of the three individual K d 

### values was within the range reported in the EPA validation program (0.685 to 1.57 nM); the mean value was above the recommended range. Although the goodness of fit (R 2 

### = 0.880-0.986) was acceptable, confidence in these numbers is low based on the large variation in Kd estimates among runs.

TABLE 5. Saturation Binding Experiment of [ 3 H]-R1881 with Androgen R eceptor from Ra t Prostate Cytosol a Parameter Run 1 Run 2 Run 3 Mean ± SE b
R 2 (unweighted) 0.880 0.986 0.893 0.880-0.986 B max (nM) 0.010 0.008 0.011 0.010±0.002 B max (fmol/100 μ g protein) 1.087 0.896 1.229 1.071±0.167 K d (nM) 0.855 0.531 1.855 1.080±0.690 a Data were obtained from supplemental inform ation provided by the laboratory (MRID 48843501). b The range of R 2 is reported and the mean ± SE is reported for the other parameters. R 2 Goodness of fit for curve calculated for specific binding.



### Figure 1 illustrates the non-specific, sp ecific, and total binding curves for [ 3 

### H]-R1881 to the androgen receptor for the three independent runs . The specific binding reached a plateau in each run, and non-specific binding was less than 20% of total binding. Figure 2 contains the Scatchard plots that illustrate the binding of [ 3 

### H]-R1881 to the androgen receptor.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None


### FIGURE 1. Binding of [ 3 

### H]-R1881 to the Androgen Receptor during the Saturation Binding Experiment.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None



### FIGURE 2. Scatchard Pl ots of the Binding of [ 3 

### H]-R1881 to the Androgen Receptor.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None



### B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive binding experiments are summarized in Table 6 and shown graphically in Figures 3-5. EPTC reduced the mean specific binding of the ligand, [ 3 

### H]-R1881, in all three runs in a concentration dependent manner. At 10 -3 

### M of EPTC, specific bi nding was reduced to 56.5, 50.0, and 50.6% in the first, second, and third assays, respectively. At 10 -4 

### M, specific binding was reduced to 89.4, 87.4, and 91.0% in the first, second and third assays, respectively. Specific binding was ≥ 92.8% at EPTC concentrations ≤ 10 -5 

### M. As specific binding was not ≤ 50% at any concentration of EPTC, an IC 50 

### and RBA could not be calculated for EPTC. All three competitive bind ing runs were classifi ed as equivocal since the percent specific [ 3 

### H]-R1881 bound to the AR was between 50 and 75% at all concentrations of EPTC tested.

The estimated log IC 50 

### s for R1881 and the weak positive co ntrol (dexamethasone) were in the ranges of -9.0 to -9.9 M and -4.4 to -4.6 M, respectively. The mean RBA for the weak positive control was 0.0016%. Confidence in these numbers is high due to the small variation. The solvent cont rol responses indicated no dr ift in the study assay.

TABLE 6. Competitive Binding Assay of EP TC with AR from Rat Prostate Cytosol a Parameter Assay 1 Assay 2 Assay 3 Mean

r 2 (unweighted) R1881 NR NR NR NR Dexamethasone NR NR NR NR EPTC NR NR NR NR Log IC 50 (M) R1881 -9.9 -9.0 -9.0 -9.3 b Dexamethasone -4.4 -4.4 -4.6 -4.5 b EPTC NA NA NA NA IC 50 (M) b R1881 1.3 x 10 -10 10 -9 10 -9 5.0 x 10 -10 Dexamethasone 4.0 x 10 -5 4.0 x 10 -5 2.5 x 10 -5 3.2 x 10 -5 EPTC NA NA NA NA RBA (as% IC 50 ) c Dexamethasone 0.0003 0.0025 0.0040 0.0016 EPTC NA NA NA NA

a Data were obtained from page 20 of the study report. b Calculated by reviewer c Calculated by reviewer: [IC 50 (in M) Dexamethasone or EPTC / IC 50 (in M) R1881] x 100% NA Not applicable NR Not reported RBA Relative binding affinityAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None


### FIGURE 3. Binding of EPTC, R188 1 and Dexamethasone in Assay 1.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None


### FIGURE 4. Binding of EPTC, R188 1 and Dexamethasone in Assay 2.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None


### FIGURE 5. Binding of EPTC, R1 881 and Dexamethasone in Assay 3.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None



### C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was functioning properly, each run was evaluated using the following criteria (Table 7):

TABLE 7. Criterion a Tolerance Limit(s) b Value Yes No Ligand depletion is minimal. The recommended ratio of total binding in the absence of competitor to total amount of [ 3 H]-R1881 added per assay tube (% binding) ≤ 15 8.7 to 9.7 X

Test chemical Top (% binding) 80 to 115 101.5 to 118.2 X R1881 fitted curve parameters Top (% binding) 82 to 114 96 to 103 X Bottom (% binding) -2.0 to 2.0 0 X Hill Slope -1.2 to -0.8 -1.0 X Weak positive control (dexamethasone) fitted curve parameters Top (% binding) 87 to 106 101 to 105 X Bottom (% binding) -12 to 12 -1 to 7 X Hill Slope -1.4 to -0.6 -1.0 to -0.6 X Saturation Binding Experiment K d (nM) 0.685 – 1.57 1.08 ± 0.690 X Non-specific binding (%) ≤ 20.0 9.7-14% X a Data were obtained from pages 23-28 of the study report. b These values represent ranges from the validation study. c Calculated by reviewer from page 34-38. Avg dpm (1 ml) R1881 (NSB) / Avg dpm (1ml) Total binding (solvent control). NR Not reported



### The performance criteria were met, with the following exceptions: in the second assay, the top specific binding (%) of EPTC was 118.2%, which slightly exceeded the range of the Guideline performance criteria (80 to 115%); and in the first run a ssay, the bottom plateau level (% binding) for dexamethasone was not ab le to be accurately calculated, because the concentration range for dexamethasone was prepar ed slightly different than the guideline in error. As these deviations were minor and were not considered to have affected the results of the study, all three runs were considered valid.

Additionally, the curve for the reference material showed that increasing concentrations of unlabeled R1881 displaced [ 3 

### H]-R1881 in a manner consiste nt with one-site binding, as indicated by a Hill slope of -1.0 to -0.6. Examination across the r uns indicated consistency of the Hill slope, placement along the X-axis, and top and bottom plateaus.

The percentage of the total specific binding in the solvent controls was approximately 10%. This was within the less than the ≤ 10-15% recommended in the guideline. Ligand depletion was also minimal. Sufficient optimi zation of the number of specific binding sites is supported curves for contro ls of acceptable steepness, low non-specific binding, and low variability.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: All three assays achieved the “equivocal” criterion described in the guide line (between 50 and 75% radio ligand binding). There wereAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 16 of 16 EPTC / 041401 OCSPP 890.1150/ OECD None


### no apparent alterations in ra diolabeled R1881 binding at any of the lower concentrations tested. EPTC was classified as “equivocal”.

B. AGENCY COMMENTS: In the saturation binding experiment, the mean Kd for [ 3 

### H]- R1881 was 1.080 nM, and the mean estimated B max 

### was 1.071 fmol/100 μg protein for the single batch of prostate cytosol that was used in the assay. On ly one of the three individual K d 

### values was within the range reported in the EPA validation program (0.685 to 1.57 nM); the mean value was above the recommended ra nge. In the competitive binding experiment, the average log IC 50 

### s for R1881 (-9.3 M) and the weak positive control, dexamethasone (-4.5 M), were within the expected ranges. The mean RBA for the weak positive control was 0.0016%.

The solvent control responses i ndicated no drift in th e study assay. The performance criteria were met, with the following exceptions: in the second assay, the top specific binding (%) of EPTC was 118.2%, which slightly exceeded the range of the Guideline performance criteria (80 to 115%); and in the first a ssay, the bottom plateau level (% binding) for dexamethasone was not able to be accurately calculated, because the concentration range for dexamethasone was prepared slightly different than the guideline in error. As these deviations were minor and were not considered to have affected the re sults of th e study, all three runs were considered valid.

EPTC reduced the mean specific binding of th e ligand in a concentration dependent manner in all three runs. The maximum binding displacement occurred at 10 -3 

### M of EPTC, with specific binding being reduced to 50-56.5%. Specific binding was also reduced at 10 -4 

### M (87.4 – 91%), but not at EPTC concentrations ≤ 10 -5 

### M ( ≥ 92.8% binding). An IC 50 

### and RBA could not be calculated for EPTC. EPTC is cl assified as equivocal in the AR assay as specific radiolabeled ligand bound was betw een 50 and 75% up to the highest EPTC concentration tested in all three runs.

C. STUDY DEFICIENCIES: The following deficiencies were noted that did not have an adverse effect on the results, interpreta tion or conclusions of this study:

 Curves were not provided showing the averag e binding of each test substance across all three runs.

 Non-specific binding exceeded the tolerance limits for Assays 2 and 3. This was not considered to have a negative impact on th e study because there was still enough of a dynamic range in which to detect changes in binding with concentr ation for control and test chemicals.

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1200; Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-13-2012 (MRID 48616402)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 12/16/2011
Secondary Reviewer Signature: Michelle Sharpe-Kass, M.S. Date: 1/17/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 1/23/2011
Quality Assurance: Signature: Jack D. Early, M.S. Date: 1/23/2011

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).EPTC/041401 Aromatase (Human Recombinant) Assay (2011) I Page 1of13 OCSPP 890.1200 I OECD None 

### Primary Reviewer: Vincent Chen M.S. 

### Signature: ___:....____::_

_____ _ Health Effects Division (7509P) Date: .2-Y :rvr-.J z..o...L.S 

### Secondary Reviewer : Greg Akerman, Ph.D. 

### Signature:

## ~/6__,.....,,,_..,,.**A'-----** Risk Assessment Branch III, Health Effects Division (7509P) Date:**--~ <i** **~/ .l** **lt )**  __ Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200



### PC CODE: 041401



### TXR: 0051762



### TEST MATERIAL (PURITY): EPTC ( > 99.9% a.i

. )



### SYNONYMS:

IUPAC: CAS: S-ethyl dipropylthiocarbamate S-ethyl N, 

### N-dipropylcarbamot hioat e 

### DP BARCODE: 0397177



### CAS No.: 759-94-4



### CITATION: Wilga
, P.C. (201 1)

. EPTC: Human Recombinant Aromatase Assay. CeeTox, Inc [4717 Campus Drive, Kalamazoo, MI 49009, US]. Study No. 9142V100349AROM. 09 November 2011. MRID 48616402. Unpublished.



### SPONSOR: Gowan Company; 370 Main St
., Yuma
, AZ 85364, US



### TEST ORDER: EDSP-041401-64



### EXECUTIVE SUMMARY: In an 

### in vitro aromatase (CYP 19) assay (MRID 48616402), EPTC ( > 99.9% w / w, Batch# Pl 105096) in dimethyl sulfoxide (DMSO) was incubated with human recombinant aromatase and tritiated androstenedione ([ 1**~- H(N)**  

### ]-androst-4-ene-3
, 17- dione ; [3H]-ASDN) at increasing logarithmic concentrations from 10

- 10 to 10

- 3 

### M for 15 minutes
at 3 7 ° C to assess the potential of EPTC to inhibit aromatase activity.

Aromatase activity was determined by mea s uring the amount of tritiated water produced at the end of a 15-minute incubation period for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Three independent runs were conducted, and each run included a full activity control, a background activity control, a positive control series (10

- 1 0 to 10

- 5 

### M) using a known inhibitor (4-hydroxyandrostenedione ; 4-0H ASDN)
, and the test chemical series c10

- 1 0 to 10

- 3 ) 

### with 3 repetitions per concentration.

Aromatase activity in the full activit y controls ranged from 0.161 to 0

. 376 nmol·mgprotein

- 1·min

- 1 for the three test runs , with a mean and standard deviation of 0.261±0.077 nmol·mg-protein

- 1 ·min

- 1

. Activity in the background controls ranged from 0.40% to 0.95% and average d 0

. 57% of the full activity controls

. The response of the full activity controls and background controls was acceptable for each run.Aromatase (Human Recombinant) A ssay (2011) / Page 2 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None 

### Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, hill slope, log IC 50 

### , and coefficient of variati on for replicates of each concentration within runs. Also, the coefficien ts of variation (%CVs) for replicates of each concentration of EPTC within a run were generally within the 15% guideline. For 4-OH ASDN, the estimated log IC 50 

### averaged –7.21 M, and the slope was -0.97.

For EPTC, aromatase activity av eraged 0.242±0.068 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentration of 10 -10 

### M and 0.213±0.011 nmol·mg-protein -1 

### ·min

− 1 

### at the highest tested concentration of 10

− 3 

### M (when Run 1 was excluded due to precipitation). The data for EPTC were not suited for modeling. The average lowest portion of the response curve across runs was between 50 and 75% activity (69% at 10 –3 

### M EPTC; excluding run 1 due to precipitation) indicating an equi vocal response.

Based on the data from the average response cu rve, EPTC is classified as Equivocal for aromatase inhibition in this assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Aromatase assay (OCSPP 890.1200).

COMPLIANCE: Signed and dated GLP Compliance, Quality Assurance, and Data Confidentiality statements were provided.Aromatase (Human Recombinant) A ssay (2011) / Page 3 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: EPTC Description: Liquid Source: Gowan Batch # (Expiration Date): P1105096 (February 4, 2015) Purity: >99.9% Vapor Pressure: 4500 mPa at 25°C Storage Conditions: Room temperature Stability: Not reported Solvent: DMSO Solubility: Visually soluble at 10

− 3 M in 2/3 test runs. Highest Concentration Tested: 10

− 3 M Stock Solution Preparation Methodology: Not reported Molecular Weight: 189.3 CAS #: 759-94-4 Structure:

N S O CH 3

CH 3 CH 3



### 2. Non-Labeled Substrate: Androstenedione (ASDN) CAS # : 63-05-8 Source: Steraloids, Inc. (Cat. # L1712) Batch # (Expiration Date): L1712 (April 2016) Purity: 99.8%



### 3. Radiolabeled Substrate: [1 β

3 

### H(N)]-Androst-4-ene-3,17-dione; ([ 3 

### H]-ASDN)
Source: Perkin Elmer (Cat. # NET-926) Batch # (Expiration Date): 619344 (January 10, 2012) Radiochemical Purity (Supplier): >97% Specific Activity: 26.3 Ci/mmol Radiochemical Purity (Vendor Determination): >97%



### 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN) CAS # 566-48-3 Source: Sigma-Aldrich (Cat. # F2552) Batch # (Expiration Date): 081K2133 (March 2015) Purity: 99.6%


### 5. Solvent (Vehicle Control): DMSO
Source: Sigma-Aldrich (Cat. # D2650) Batch # (Expiration Date): RNBB7886 (February 2013) Justification for Choice of Solvent: Acceptable vehicle by OCSPP guideline 890.1200 Concentration (% of Total Volume in Assays): 1% v/vAromatase (Human Recombinant) A ssay (2011) / Page 4 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None



### 6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes
Source: BD Biosciences Gentest TM (Woburn, MA; Cat. # 456260) Lot # (Expiration Date): 03897 (February 2014) Protein Concentration: 7.4 mg/mL Cytochrome C Reduc tase Activity: 290 nmol/min/mg Aromatase Activity: 6.0 pmol/min/pmol P450



### B. METHODS

1. Assay Components and Preparations: A mixture of non-labe led and radiolabeled [ 3 

### H]ASDN was prepared such that the final concentration of ASDN in the assay was approximately 0.1 μM, and the amount of tri tium added to each incubation tube was 0.1 μ Ci.

Test chemical(s) stock solutions were prepar ed such that the total volume of each test chemical formulation used per assay was no more than 1% v/v of the total assay volume to minimize the potential for the solvent to inhi bit the enzyme. The report specified that DMSO was chosen over ethanol as the solv ent of choice for the following reasons: 1) DMSO was listed as one of the vehicles ac ceptable for use in OC SPP 890.1200 guideline; 2) DMSO was not as volatile as ethanol and so ev aporation was less of a concern in the assay, and 3) DMSO was more accurate to pipette because of density and viscosity.

A stock solution of the positive control substance, 4-OH ASDN, was formulated in DMSO. Fresh dilutions of the stock solution were prepar ed in the same solvent as the stock solution on the day of use. Dilutions were prepared such that the target concentrations of the positive control substance (10 -10 

### to 10 -5 

### M; Table 4) were achieved by the addition of 20 μL of the dilution for a final assay volume of 2 mL.

Human recombinant microsomes were purchased from BD Biosciences, and stored at -80 ± 10oC for no longer than 7 months for this study. Before use of the microsomes, the batch was thawed and sub-aliquoted at 0.008 mg/mL into individual vi als before being refrozen to minimize freeze-thaw cycles to no more than one.

Other assay components sodium phosphate buffer, propylene glycol, and NADPH are reported in Table 1.

TABLE 1. Assay Components and Conditions Assay Factor Values Sodium phosphate buffer (pH 7.4) 0.1M Microsomal protein 0.004 mg/mL NADPH 0.3 mM [ 3 H]ASDN 100 nM Propylene glycol 5% Temperature 37±2°C Incubation time 15 min


### 2. Suitability Assessments: The protein concentration of the microsomes was supplied by the vendor as 7.4 mg/mL. Aromatase activity of the microsomes was also provided by the vendor as 6.0 pmol/min/pmole P450. The minimum aromatase activity in the full activityAromatase (Human Recombinant) A ssay (2011) / Page 5 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None 

### control samples was determined to be 0.161 nm ol/min/mg protein, which was greater than the minimum recommended aromatase ac tivity of 0.1 nmol/mg-protein/min.

3. Aromatase Assay: Each assay run contained 4 tube s for the full enzyme activity and 4 tubes for the background activity controls. Two tubes of each control were run at the beginning of the assay, and two of each control were run at the end of the assay. A full concentration curve in duplicate for the positiv e control, and a full concentration curve in triplicate for the test substa nce were established. The aromatase assay was conducted generally according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

### H 2 

### O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of nmol·mg-protein -1 

### ·min -1



### . 4. Demonstration of Proficiency: No information was provided c oncerning demonstration of proficiency. However, the report stated that prior to conducting this assay using EPTC, a full-scale assay consisting of three indepe ndent runs was conducte d using the positive control (4-OH ASDN), econazole, fenarimol, nitrofen, and atrazine. Proficiency was demonstrated when the positive control met the performance criteria from the guideline and by the correct classification of the four profic iency chemicals in Table 6 of the guideline. Data were generated by CeeTox a nd are maintained at CeeTox.

a. Positive Control: Data were not provided.

(1) Initial Demonstration of Laboratory Proficiency: The positive control data (new/historical data for laborator y) were not reported in rega rds to the following criteria:

 Mean aromatase activity in the absence of an inhibito r was at least 0.1 nmol/mgprotein/min.

 Mean background control activity was ≤ 15% of the full activity control.

 Coefficient of variation (% CV) for repl icates within each sample type and concentration of 4-OH ASDN was <15%.

Performance criteria were reported for the test runs only (Table 2), and served as guidance in identifying runs that provided parameters in the preferred ranges.

(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): Demonstration of proficiency by a ne w technician, if applicable, was not reported. The positive control data for slope, top and bottom percent, and log IC 50 

### met the criteria as listed in section (i) of OCSPP 890.1200 with the fo llowing exceptions: (i) the top percent for one run (run 2) was slightly be low (88.48%) the recomme nded range; and (ii) the log IC 50 

### for one run (run 3) was slightly below (-7.35) the recommended range.

TABLE 2. Performance Criteria for the Positive Control a Parameter Lower Limit Criteria Upper Limit Criteria Actual Lower Limit Actual Upper Limit Slope -1.2 -0.8 -1.02 -0.94 Top (%) 90 110 88.48 104.31 Bottom (%) -5 +6 -0.28 0.66 Log IC 50 (M) -7.3 -7.0 -7.35 -7.09Aromatase (Human Recombinant) A ssay (2011) / Page 6 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None

a Data were obtained from page 27 of the study report.



### b. Proficiency Chemicals: Data not provided.

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations Econazole 24169-02-6 Inhibitor Not reported Fenarimol 60168-88-9 Inhibitor Not reported Nitrofen 1836-75-5 Inhibitor Not reported Atrazine 1912-24-9 Non-inhibitor Not reported



### 5. Determination of Aromatase Ac tivity with Test Chemical: The response of aromatase activity to the presence of eight concentrations of EPTC per run, in triplicate, was tested during three independent runs. Solubility was visually assess ed (presence of cloudiness or a precipitate). The test compound was soluble at 10 –3 

### M in two runs, but a slight amount of precipitation was visible in the firs t run. The full enzymatic activity ( ≥ 95% for the means of each run) was obtained at the two lowest con centrations of EPTC, defining the top of the concentration-response curve.

TABLE 4. Test Chemical Study Design for Each Test Run Sample Type Repetitions (Tubes) Description Reference or Chemical (M) Full Activity Control 4 All test components a plus solvent vehicle N/A Bkgd Activity Control 4 Same as above without NADPH N/A 4-OH ASDN Conc 1 2 All test components plus 4-OH ASDN 1×10 –5 4-OH ASDN Conc 2 2 All test components plus 4-OH ASDN 1×10 –6 4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10 –6.5 4-OH ASDN Conc 4 2 All test components plus 4-OH ASDN 1×10 –7 4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10 –7.5 4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10 –8 4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10 –9 4-OH ASDN Conc 8 2 All test components plus 4-OH ASDN 1×10 –10 EPTC Conc 1 3 All test components plus EPTC 1×10 –3 EPTC Conc 2 3 All test components plus EPTC 1×10 –4 EPTC Conc 3 3 All test components plus EPTC 1×10 –5 EPTC Conc 4 3 All test components plus EPTC 1×10 –6 EPTC Conc 5 3 All test components plus EPTC 1×10 –7 EPTC Conc 6 3 All test components plus EPTC 1×10 –8 EPTC Conc 7 3 All test components plus EPTC 1×10 –9 EPTC Conc 8 3 All test components plus EPTC 1×10 –10

a The complete assay contained buffer, pr opylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH.



### C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatas e activity (nmol/mg protein/min) and percent control for the positive control and test chemical. The following raw data and calculated endpoints for each run were in cluded in the report (Table 5).Aromatase (Human Recombinant) A ssay (2011) / Page 7 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None

TABLE 5. Raw and Calculated Data Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed aqueous incubation mixture X Average DPM/mL for each aqueous portion (after extraction) X Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X Total DPM after extraction corrected for background X Aromatase activity expressed in nmol/mg protein/min X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations X DPM Disintegrations per minute



### 2. Statistical Methods: Statistical analyses and graphica l displays were conducted using IDBS XLfit v5.2.0.0 (Guildford, UK). Basic stat istical analyses were performed on the data, which included means of replicates, standa rd deviation of the mean, standard error of the mean, and coefficient of variation. Anal ysis of variance (ANOVA) was not performed.

The response curve was fitted by weighted leas t squares nonlinear regression analysis with weights equal to 1/Y. Model fits were carried out using a 4-parameter regression model (XLfit v5.2.0.0, Fit Model 208) and Tukey’s Bi-Wei ght statistical analysis for outlier analysis. For each run, percent of control activity were plotted versus logarithm (base 10) of the EPTC concentration. Each run was plotted wi th the data’s best fit curve. Additionally, the average inhibition response curve across all runs was also plotted.

3. Interpretation of Results: Interpretation of the assay results was based on the average of three runs, using the categor ies presented in Table 6.

TABLE 6. Interpret ation of Results Criteria Interpretation Data fit 4-parameter nonlinear regression model Average curve across runs crossed 50% a Inhibitor Average lowest portion of curves across runs is between 50% and 75% activity
b Equivocal

Average lowest portion of curves across runs is greater than 75% activity b Non-inhibitor Data do not fit model ---

a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near -1. Unusually steep curves may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than -2.0, the result is classified as equivocal.
b If the test compound was not soluble above 10 -6 M and the inhibition curve does not cross 50%, the chemical is typically determined to be untestabl e in the aromatase assay.



### II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.161 to 0.376 nmol·mg-protein

− 1 

### ·min

− 1 

### for the three test runs, with a mean and standard deviation of 0.261 ± 0.077 nmol·mg-protein

− 1 

### ·min

− 1 

### . Activity in the background controls ranged from 0.40% to 0.95% and averaged 0.57% of the full activity controls. The response of each full activity control within a run was between 90 to 110% of the average full activity.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.257 ± 0.078 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentrationAromatase (Human Recombinant) A ssay (2011) / Page 8 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None 

### (10

− 10 

### M) and 0.002 ± 0.001 nmol·mg-protein

− 1 

### ·min

− 1 

### at the highest tested concentration (10

− 5 

### M). The mean aromatase activity of the positive control (expressed as % full control activity) for each concentration tested across a ll three runs is presented in Table 7, along with the overall standard deviation, standard error of the mean, and %CV. Inhibition response curves for the positive control from each run are shown in Figure 1. These results were generally within the recommended ranges for the top of the curve, bottom curve, hill slope, log IC 50 

### , and coefficient of variation for replicat es of each concentra tion within runs. The following minor exceptions were observed: in Run 2 of the assay, the "top of curve" value (e.g., 88.48%) was marginally lower than the specified range compared to the guideline range of 90%-110%; in Run 3 of the assay, the IC 50 

### was marginally lower than the specified range (log IC 50 

### = -7.35) compared to the guideli ne range of -7.3 to -7.0. The coefficient of variations (CVs) for replicates within each concentration of 4-OH ASDN and EPTC were generally within the 15% guideline, with exceptions being 18.7%, 16.1%, 23.6%, and 18.8%.

TABLE 7. Effect of EPTC on Aromatase Activity (as Percent of Control) from Independent Runs a Chemical Conc. (Log M) # Runs Overall Mean Overall SD Overall SEM Overall %CV 4-OH ASDN (positive control) -5 3 0.83 0.09 0.04 11.3 -6 3 6.53 0.89 0.36 13.6 -6.5 3 16.91 2.34 0.96 13.8 -7 3 36.66 2.76 1.13 7.5 -7.5 3 64.65 3.60 1.47 5.6 -8 3 81.24 5.06 2.07 6.2 -9 3 93.92 9.84 4.02 10.5 -10 3 98.58 8.83 3.61 9.0 EPTC -3 3 69.32 5.54 2.26 8.0 -4 3 96.53 6.40 2.61 6.6 -5 3 100.17 7.98 3.23 7.9 -6 3 94.33 8.75 3.57 9.3 -7 3 98.90 9.51 3.88 9.6 -8 3 92.24 10.32 4.21 11.2 -9 3 95.23 8.84 3.61 9.3 -10 3 93.63 12.49 5.10 13.3

a Values were calculated by the reviewers based on data provided on pages 41-54. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of VarianceAromatase (Human Recombinant) Assay (2011) / Page 9 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None 

### FIGURE 1. Inhibition Respon se Curves for 4-OH ASDN.

C. TEST SUBSTANCE: For EPTC, aromatase activity averaged 0.242 ± 0.068 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentration of 10 -10 

### M and 0.213 ± 0.011 nmol·mg-protein -1 

### ·min

− 1 

### at the highest tested concentration of 10

− 3 

### M (when Run 1 wasAromatase (Human Recombinant) A ssay (2011) / Page 10 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None 

### excluded due to precipitation). Precipitation was only observed in Run 1 at 10

− 3 

### M, and the report stated that the microsomal reaction “appeared to receive no NADPH.” The mean aromatase activity of EPTC (expressed as % full control activity) for each concentration tested across all three runs is provided in Table 7 (presented above), along with the overall standard deviation, standard error of the mea n, and %CV. Inhibition response curves for EPTC from each run are shown in Figure 2, and the average inhibition response curve across all runs is shown in Figure 3.Aromatase (Human Recombinant) A ssay (2011) / Page 11 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None 

### FIGURE 2. Inhibition Response Curves for EPTC.Aromatase (Human Recombinant) A ssay (2011) / Page 12 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None 

### FIGURE 3. Mean Inhibition Response Curve for EPTC.

The data for EPTC were not suitable for m odeling. Although the mean aromatase activity of 10 –3 

### M EPTC was determined to be 69.3% of control activity (excluding Run 1 due to precipitation), the best fit mean inhibition re sponse curve (Figure 3) was a straight line (essentially no inhibition was observed at other doses). The effect of the positive control on inhibition of aromatase activity is presented in Table 8. For 4-OH ASDN, the estimated log IC 50 

### averaged –7.21 M, and the slope was -0.97. Confidence in these values is very high due to the small variation (<5% CV).

TABLE 8. Effect of EPTC and 4-OH ASDN on Ar omatase Activity (as Percent of Control) from Independent Runs a Chemical Run 1 Run 2 Run 3 Mean SD %CV Log IC 50 (M) EPTC NA NA NA NA NA NA 4-OH ASDN -7.19 -7.09 -7.35 -7.21 0.13 1.82 Slope EPTC NA NA NA NA NA NA 4-OH ASDN -0.94 -1.02 -0.95 -0.97 0.04 4.49 a Data were provided on page 27 of the study report. M ean, SD, and %CV were calculated by the reviewers. SD Standard Deviation CV Coefficient of Variance NA Not applicable. Values for EP TC were not suited for modeling.


### As aromatase activity was only 69.3% of cont rol activity at the highest dose, EPTC was determined to be “equivocal” for inhibiti on of aromatase activity in this assay.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: EPTC was determined to be “equivocal” for inhibition of aromatase activity in this assay.Aromatase (Human Recombinant) A ssay (2011) / Page 13 of 13 EPTC / 041401 OCSPP 890.1200 / OECD None


### B. AGENCY COMMENTS: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.257 ± 0.078 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentration (10

− 10 

### M) and 0.002 ± 0.001 nmol·mg-protein

− 1 

### ·min

− 1 

### at the highest tested concentration (10

− 5 

### M). These results were generally within the recommended ranges for the top of the curve, bottom curve, hill slope, log IC 50 

### , and coefficient of varia tion for replicates of each concentration within runs. The %CVs for re plicates within each concentration of 4-OH ASDN and EPTC were generally within the 15% guideline. Minor deviations from the guideline ranges did not confound the results of this study.

For EPTC, aromatase activity aver aged 0.242 ± 0.068 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentration of 10 -10 

### M and 0.213 ± 0.011 nmol·mg-protein -1 

### ·min

− 1 

### at the highest tested concentration of 10

− 3 

### M (Run 1 was excluded due to precipitation). Precipitation was only observed in Run 1 at 10

− 3 

### M. As precipitation was obser ved in the first run at the highest dose, it is unclear why further evaluations were not carried out at 10 –3.5 

### M. It is also unclear why precipitation at 10

− 3 

### M was only observed in Run 1. For 4-OH ASDN, the estimated log IC 50 

### averaged –7.27 M, and the slope was -1.00. Confidence in these values is very high due to the small variation (<5% CV).

The data for EPTC were not suitable for m odeling. Although the mean aromatase activity of 10 –3 

### M EPTC was determined to be 69.3% of control activity (excluding Run 1 due to precipitation. As the average lowest portion of curves across runs was between 50% and 75% activity (69.3%), EPTC was determined to be “equivocal” for inhibition of aromatase activity in this assay.

C. STUDY DEFICIENCIES: The following deficiency was note d that is not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 Proficiency chemical da ta were not provided.

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-13-2012 (MRID 48616404)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Michelle Sharpe-Kass, M.S. Date: 1/10/2012

Secondary Reviewer Signature: David A. McEwen, B.S. Date: 1/23/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 1/25/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 1/25/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).EPTC/ 04140 I Estrogen Receptor Binding (Rat Uterine Cytosol) (20 t I) I Page I of 17 OCSPP 890.1250/ OECD None 

### Primary Reviewer: Sheila Healy, Ph

. D. 

### Health Effects Division Secondary Reviewer: Greg Akerman, Ph

. D. 

### Health Effects Division

DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC); OCSPP 890.1250



### PC CODE: 041401 

### DP BARCODE: D397177



### TXR#: 0051762 

### CAS No.: 759-94-4



### TEST MATERIAL (PURITY): EPTC (99.9% a.i.)



### SYNONYMS: S-Ethyl dipropylthiocarbamate



### CITATION: Willoughby, J.A.
, Sr. (2011). EPTC: Estrogen Receptor Binding (Rat Uterine Cytosol). CeeTox, Inc.
, Kalamazoo
, MI. Laboratory Study Number : 9142V100349ERB
, November 10
, 2011. MRID 48616404. Unpublished.

Willoughby, J .A. (2012). Supplemental Information

- Laboratory Proficiency Data for ERTA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo
, MI. Julyl 9, 2012. MRID 48843501. Unpublished.



### SPONSOR: Gowan Company, 370 Main Street
, Yuma
, AZ



### TEST ORDER#: EDSP-041401-64



### EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48616404), uterine cytosol from Sprague-Dawley rats was used as the source of estrogen receptors (ER) to conduct binding experiments. A competitive binding experiment was conducted to measure the binding of a single concentration of [3H]- l**7~-estradiol** 

### ( 1 nM) in the presence of increasing concentrations of EPTC (logarithmic increase from 10

- 10 

### to 10

- 3 

### M)

. DMSO was used as a vehicle at a final concentration of 4%

. The assay included 19-norethindrone as a weak positive control
, octyltriethoxysilane as a negative control, and**17-~-estradiol** 

### as the natural ligand reference material.

Saturation binding data were not originally provided in the study report; however
, summarized
sat uration binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The protein concentrations used in the saturation binding

runs varied between each run , and were approximately 3- to 6-fold greater than recommended (160 to 320 μg versus 50± 10 μg)

. The mean dissociation constant (Kct) for**[3H]-17~-estradiol** 

### was 0.331±0.061 nM and the estimated Bmax was 74.55 ± 3

. 03 fmol/100 μg protein for theEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None


### prepared rat uterine cytosol. The K d 

### for each run was within the expected Guideline range of 0.03 to 1.5 nM.
In the competitive binding experiment, the mean IC 50 

### was 9.3 x 10 − 10 

### M for the natural ligand, 17 β -estradiol, and 3.2 × 10

− 6 

### M for the weak positive control, which had a relative binding affinity (RBA) of 0.029%. Performance criteria we re met with the exception of the Hill slope for 17 β -estradiol was outside the recommended range (

− 1.1 to

− 0.7) two for the runs (

− 1.4 and

− 1.7, respectively and the bottom plateau of the binding curve for 17 β -estradiol for run 3 (5%) exceeded the recommended range (

− 4 to 1%).

An estimated mean IC 50 

### was not calculated for EPTC as it did not displace 50% or the radiolabeled ligand from the ER. In two assay runs, EPTC reduc ed the specific binding of the radiolabeled estradiol at the hi ghest concentration tested (10

− 3 

### M) to 96-107%; in Run 3 percent binding was 69.6% at 10

− 3 

### M. Based on the results from the three runs, EPTC is classified as Not Interactive in the Estrogen Receptor Binding Assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Estrogen Receptor Binding Assay (OCSPP 890.1250).

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc.
Location: Kalamazoo, MI Study Director: Jamin A. Willoughby, Sr., Ph.D Other Personnel: Karen Rutherford, Director of Laboratory Operations David Blakeman, Senior Scientist/Endocrine Group Leader Cameron Haines, Scientist Steven McColley, Scientist Colleen Toole, Director of Project Management Study Period: May 25, 2011 - November 10, 2011



### 2. Test substance: EPTC Description: Liquid Source: Gowan Company Lot/Batch #: P1105096, Exp. Feb 4, 2015 Purity: 99.9 % w/w Solubility: Not reported (NR) Volatility: NR Stability: NR Storage conditions: Ambient CAS #: 759-94-4 Molecular weight: 189.3 g/mol Structure:

N S O CH

3

CH 3 CH 3



### 3. Non-labeled ligand: 17 β -estradiol Supplier: Sigma-Aldrich, St Louis, MO Catalog # E8875 Batch #: 110M0138V Purity: 100% CAS #: 50-28-2


### 4. Radioactive ligand: [ 3 

### H]-17 β -estradiol Supplier: Perkin-Elmer, Boston, MA Catalog #: NET517 Batch#: 650702 Radiochemical purity: NR Specific activity: 130.2 Ci/mol Concentration of stock: 1 nM



### 5. Positive control: 19-norethindrone Supplier: Sigma-Aldrich, St Louis, MO Catalog # N4128 Batch #: 030M1359V Purity: 99% CAS # : 68-22-4


### 6. Negative control: Octyltriethoxysilane Supplier: Sigma-Aldrich, St Louis, MOEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None

Catalog # 440213 Batch #: 24996KK Purity: 99.34% CAS # : 2943-75-1



### 7. Solvent/vehicle control: DMSO
Justification for choice of solvent: Recommended solvent according to guidel ine, and EPTC is soluble up to 10 -3 M Final Concentration: 4%



### B. METHODS

1. Preparation of Rat Uterine Cytosol (RUC): Rat uterine tissue was obtained from Harlan laboratories, from female Sprague-Dawley rats that were ovariectomi zed 7 days prior to being euthanized. Animals were 12-13 weeks old at the time of euthanasia. The uteri were frozen and shipped overnight on dry ice. Once at the lab, uteri were inspected for signs of residual ovarian tissue, weighe d, placed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffe r and used immediately. Uteri were homogenized in buffer, centrifuged for 10 min at 2500 × g at 4 oC. The resulting supernatant was transferred and centrif uged for 60 minutes at 105,000 × g , discarding the resulting pellets. Protein concentrati on of the cytosol was determined to be 3.22 mg/mL using a protein kit compatible with DTT in the TE DG buffer (e.g., BioRad Protein Assay Kit). Cytosol was divided into ali quots (1-6 mL) for immediate use or storage at -80 oC.

2. Saturation (Radioligand) Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [ 3 

### H]-17 β -estradiol was performed to demonstrate that the ER was present in reasonable concentr ations and had the appropriate affinity for the native ligand (MRID 48843501). The conditions fo r the saturation binding are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a Source of receptor Rat uterine cytosol Concentration of radioligand (as serial dilutions) 0.03 to 3 nM Concentration of non-labeled ligand (100X [radioligand]) 3 to 300 nM Concentration of receptor Sufficient to bind approximately 25 to 35% of radioligand at 0.03 nM Temperature 4oC Incubation time 16 to 20 hours Composition of assay buffer Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride (PMSF) 1 mM DTT 1 mM a Data were obtained from page 1 of the study report (MRID 48843501).



### On the day of each assay, the specif ic activity of the stock solution [ 3 

### H]-17 β -estradiol (originally 130.2 Ci/mmol as manufactured on May 6, 2011) was adjusted for decay over time (adjusted specific activities were not re ported), and serial dilutions in TEDG+PMSF buffer were prepared to achieve the fi nal concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1,Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None



### and 3 nM. Solutions of non-labeled 17 β -estradiol were prepared in a similar manner to achieve concentrations that were 100-fold greater than each re spective radiolabeled concentration to resu lt in final concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM.

For each batch of cytosol, the optimal protein concentration was determined by testing serial amounts of protein per tube, using 0.03 nM radi olabeled estradiol, until a concentration was reached that bound approximately 25 to 35% of the total radioactivity added. The final protein concentrations were 320 μg, 192 μg and 160 μg per assay tube for the first, second and third saturation binding e xperiments, respectively ( Note: typically 50 ± 10 μg protein per tube) . Each assay consisted of three non- concurrent runs (conducted on August 5, 6, and 7, 2011, respectively). Each run included three replicates of each test substance at each concentration, resulting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a Total binding b Non-specific binding c Radioligand alone d Assay Components

Tubes 1-24 Tubes 25-48 Tubes 49-72 350 μL 300 μL --- TEDG+PMSF buffer 50 μL 50 μL 50 μL [ 3 H]-17 β -estradiol (8 serial dilutions) e

--- 50 μL --- Non-labeled 17 β -estradiol (8 serial dilutions, 100x each respective labeled concentration) f 100 μL 100 μL --- Uterine cytosol (diluted to appropriate concentration) 500 μL 500 μL 50 μL Total volume in each assay tube a Data were obtained from page 2 of the study report (MRID 48843501). b Total binding = [ 3 H]-17 β -estradiol bound to ER c Non-specific binding = [ 3 H]-17 β -estradiol and 100-fold greater non-labeled bound to ER d Total [ 3 H]-17 β -estradiol alone for dpm determination at each concentration e Final concentrations of [ 3 H]-17 β -estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. f Final concentrations of non-labeled 17 β -estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



### Tubes were incubated with gentle vort exing for 17.5-19 hours at approximately 4  C. To separate bound from free estradiol, hydroxyapati te (HAP) slurry was added to each tube and vortexed (3 times with 5-minute intervals). S ubsequently, the contents of each tube were washed three times as follows: 2-mL portions of TEDG+PMSF buffer were added, vortexed, centrifuged fo r 10 minutes at 1000 x g , and the supernatant decanted and discarded. After the final centrifugation, et hanol (1.5 mL) was added to the HAP pellet remaining in each tube to extract the [ 3 

### H]-17 β -estradiol, followed by vortexing, and centrifugation for 10 minutes at 1000 x g . Aliquots (1 mL) of supernatant were radioassayed by scintillation counting. The te mperature was maintain ed at approximately 4oC throughout the assay prior to extraction with ethanol.

3. Competitive Binding Experiment: A summary of the experiment conditions for the competitive binding experiment is included in Table 3.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None

TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat Uterine Cytosol Concentration of radioligand 1 nM Concentration of receptor Sufficient to bind 10-15% of radioligand b Concentration of test substance (as serial dilutions) 10

− 10 to 10

− 3 mM Temperature 4 ± 2  C Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4)
EDTA 1.5 mM Glycerol 10% (v/v) Protease Inhibitor 0.5% (v/v)
DTT 1 mM a Data were obtained from page 15 of the study report. b Calculated by reviewer from data on pages 35, 38 and 41



### A solubility test was performed to determine the appropriate solvent (DMSO) using visual observation to examine for precipitation (use of magnification as not reported). On the day of the assay, the specific activity of the stock solution [ 3 

### H]-17 β -estradiol was adjusted for decay over time, and diluted in TEDG + PMSF buffer to achieve a final concentration of 1 nM. For each batch of cytosol, the optim al protein concentration was determined by testing serial amounts of prot ein per tube, using 1.0 nM ra diolabeled estradiol, until a concentration was reached that bound 10-15% of the total radioactivity added (80 μg protein per tube). Serial dilutions of EPTC, the weak positive control (19-norethindrone), the negative control (octyltriethoxysilane), a nd the reference material (non-labeled 17 β -estradiol) were prepared to achieve the c oncentrations shown in Table 4. Each assay consisted of three runs, and each run contained three replicates at each concentration, six replicates measuring total activity (50 μ L master mix) plus three measuring to tal binding (solvent control) resulting in a total of 306 samples.

TABLE 4. Molar (M) concentrations in Competitive Binding Assay Run a b

EPTC Weak Positive control Negative control Reference Chemical 19-Norethindrone Octyltriethoxysilane Non-labeled 17 β -estradiol Tubes 79-102 Tubes 31-54 Tubes 55-78 Tubes 1-30 10

− 10 10

− 8.5 10

− 10 Solvent control/master mix c 10

− 9 10

− 7.5 10

− 9 10

− 11 10

− 8 10

− 7 10

− 8 10

− 10
10

− 7 10

− 6.5 10

− 7 10

− 9.5
10

− 6 10

− 6 10

− 6 10

− 9
10

− 5 10

− 5.5 10

− 5 10

− 8.5
10

− 4 10

− 4.5 10

− 4 10

− 8
10

− 3 10

− 4 10

− 3 10

− 7 a Data were obtained from pages 24, 26, 28, and 35-43 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None

b Each tube contains: 10μL of either the test substance, positive control, negative control, solvent control, or non-labeled 17 β -estradiol; 390 μL of TEDG + PI buffer with [ 3 H]-17 β -estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL. c Solvent is DMSO in Tubes 7-9.



### Tubes were incubated with gen tle vortexing for 16-20 hours at 4 ± 2  C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and the tubes were vortexed (3 times with 5-minute intervals). S ubsequently, the contents of each tube were washed three times as follows: TEDG+PI buffer was added, vortexed, centrifuged for 10 min at 1000 × g , and the supernatant decanted and dis carded. Ethanol was added to the HAP pellet remaining in each tube to extract the [ 3 

### H]-17 β -estradiol, allowed to sit at room temperature for 15-20 min with vortexi ng, and centrifugation for 10 min at 1000× g . An aliquot of supernatant was radioassayed by scintillation counting. The temperature was maintained at 4 ± 2 oC throughout the assay prior to extraction with ethanol.

C. DATA ANALYSIS: For the saturation binding experi ment, total binding and non-specific binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 (GraphPad Software, Inc., La Jolla, CA), incorporating automatic outlier elimination according to the method of Motulsky and Brown (2006) 1 

### implemented by using the ROUT procedure in Prism v. 5 

### with a Q value of 1.0. Receptor binding data plots were corrected for ligand depletion with the method of 

### Swillens (1995) 2 

### . For the competitive binding assay, GraphPad Prism was used to generate nonlin ear regression used to fit a curve (for 17 β

estradiol, the positive control, and the test substance) to the Hill equation formula, which incorporated IC 50 

### as a parameter to be estimated. For parameters reported from the competitive binding experiment (log IC 50 

### and RBA), mean and standard deviation were calculated for each run and mean and standard e rror were calculated for the composite three runs using XLfit.

1. Definitions

a. Classification of test material: Classification of the test mate rial is based on the average of three runs. Each run was first in dividually classified as follows:

Interactive = lowest point on the fitted curve within the range of the data is less than 50% (i.e., >50% of the radiolabeled estrad iol has been displaced from the ER).

Not interactive = there are usable data points at or above 10

− 6 

### M and either the lowest point on the fitted response curve within the range of the data is above75% (i.e., <25% of the radiolabeled estradiol has been displaced from the ER) or a binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%.

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outlie rs when fitting data with nonlinear regression: a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics , Vol 7, pp 123-142. 2 Swillens, S. (1995) Interpretation of binding curves obtain ed with high receptor concentrations: practical aid for computer analysis. Molec. Pharmacol. 47(6):1197-1203.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None


### Equivocal up to the limit of concentrations tested = there are no data points at or above a test chemical concentration of 10 -6 

### M and either a binding curve can be fit but ≤ 50% of the radiolabeled estradiol has b een displaced from the ER or a binding curve cannot be fit and the lowe st average per cent binding among concentration groups in the data is >50%.

Equivocal = A run is classified as equivocal if it does not fall into any of the categories above.

The categorical classification of each run was assigned a numerical value as follows:

Run Classification Numerical Value Interactive 2 Equivocal 1 Not interactive 0 Equivocal up to the limit of concentrations tested “missing”

The values for each run were then averaged acro ss runs and the chemical classified using the following ranges:

Test Material Classification Numerical Range Interactive average ≥ 1.5 Equivocal 0.5 ≥ average <1.5 Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing”

b. Descriptors for receptor binding:

B max 

### : maximum specific binding number (fmol ER/1 00 μg cytosol protein) measures the concentration of active receptor sites K d 

### : dissociation constant (nM), measures the a ffinity of the receptor for its natural ligand IC 50 

### : concentration of the test substance at whic h 50% of the radioligand is displaced from the receptor Relative Binding Affinity (RBA %): IC 50 

### of 17 β -estradiol ÷ IC 50 

### of test substance × 100

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5. The mean K d 

### for [ 3 

### H]-17 β -estradiol was 0.331±0.061 nM, and the estimated B max 

### was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM. Although the Scatchard plots fit st raight lines to the data, the concavity observed in the data sets may indicat e issues with ligand depletion.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None

TABLE 5. Saturation Binding Experiment of [ 3 H]-17 β -estradiol with Estrogen Receptor from Rat Uterine Cytosol a Parameter Run 1 Run 2 Run 3 Mean ± SE b
R 2 (unweighted) 0.976 0.982 0.980 0.976-0.982 B max (nM) 0.148 0.102 0.080 0.110±0.035 B max (fmol/100 μ g protein) 69.25 79.76 74.65 74.55±3.03 K d (nM) 0.453 0.286 0.255 0.331±0.061 a Data were obtained from page 3 of the study report (MRID 48843501). b The range of R 2 is reported and the mean ± SEM is reported for the other parameters. R 2 Goodness of fit for curve calculated for specific binding.



### Figure 1 illustrates the non-specific, sp ecific, and total binding curves for [ 3 

### H]-17 β -estradiol to the ER for the three independent runs. The spec ific binding reached a plateau in each run, and non-specific binding was less than 20% of total bi nding. Figure 2 contains the Scatchard plots that illustrate the binding of [ 3 

### H]- 17 β -estradiol to the ER.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None


### FIGURE 1. Binding of [ 3 

### H]-17 β -estradiol to the Estrogen R eceptor during the Saturation Binding Experiment.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None



### FIGURE 2. Scatchard Pl ots of the Binding of [ 3 

### H]-17 β -estradiol to the Estrogen Receptor.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None



### B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive binding experiments are summarized in Table 6 and presented graphica lly in Figures 3-5. EPTC had no effect on specific binding at concentrations of ≤ 10 − 3 

### M in Run 2 and at ≤ 10 − 4 

### in Runs 1 and 3. However, EPTC at 10 -3 

### M reduced specific binding slightly in Run 1 (96.2%) and by 69.6% in Run 3. The estimated mean log IC 50 

### was not calculated for EPTC as the percent displacement of labeled ligand binding did not reach 50% for any run. The mean IC 50 

### for the positive control was 3.2 × 10

− 6 

### M. The mean RBA was 0.029% for the weak positive control, and a RBA could not be calculated for EPTC.

As EPTC displaced <25% of the radiolabel ed estradiol at concentrations up to 10

− 3 

### M in two runs (score of 0) and between 25 and 50% in the third run (score of 1), the overall classification for EPTC is not interactive (mean sc ore of 0.3) in this assay (Table 7).

TABLE 6. Competitive Binding Assa y of EPTC with Estrogen Rece ptor from Rat Uterine Cytosol a Parameter Run 1 b Run 2 b Run 3 b Mean

r 2 (unweighted), 17 β -estradiol NR NR NR NR Positive control NR NR NR NR EPTC NR NR NR NR Log IC 50 (M), 17 β -estradiol

− 9.0

− 9.1

− 9.0

− 9.0 19-norethindrone

− 5.6

− 5.4

− 5.5

− 5.5 EPTC NA NA NA NA IC 50 (M), 17 β -estradiol 1 x 10

− 9 7.9 x 10

− 10 1 x 10

− 9 9.3 x 10

− 10 19-norethindrone 2.5 x 10

− 6 4.0 x 10

− 6 3.2 x 10

− 6 3.2 x 10

− 6 EPTC NA NA NA NA Log (RBA) 19-norethindrone

− 1.4

− 1.7

− 1.5

− 1.5 EPTC NA NA NA NA RBA (%), 19-norethindrone 0.040 0.020 0.032 0.029 EPTC NA NA NA NA a Data were obtained from page 21 of the study report. IC 50 , RBA, and Log (RBA) were calculated by reviewer.

r 2 Goodness of fit RBA Relative binding affinity NR Not reported NA Not applicable

TABLE 7. Binding Classification of EPTC with Estr ogen Receptor a Run 1 2 3 Mean c Binding Classification d Classification category value b 0 0 1 0.3 Not Interactive a Data were calculated by the reviewer. b Classification category value: Interac tive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean). c Mean of three runs expressed to the tenths place d Interactive = mean ≥ 1.5; Equivocal = 0.5 ≤ mean <1.5; Not inte ractive = mean <0.5Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None


### FIGURE 3. Percentage [3H]-E2 Bound to th e Estrogen Receptor in the Presence of EPTC, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 1Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None


### FIGURE 4. Percentage [ 3 

### H]-E2 Bound to the Estrogen R eceptor in the Presence of EPTC, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 2Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None


### FIGURE 5. Percentage [3H]-E2 Bound to th e Estrogen Receptor in the Presence of ETPC, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 3Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None



### C. PERFORMANCE CRITERIA: To ensure that the compe titive binding assay functioned properly, each run was evaluated using the following criteria shown in Table 8:

TABLE 8. Performance Criterion a Tolerance Limit(s) Value Yes No

17 β -estradiol fitted curve parameters Log e residual SD ≤ 2.35 1.20-1.95 X Top (% binding) 94 to 111 95-109 X Bottom (% binding)

− 4 to 1

− 1 to 5 X Hill Slope (log 10 (M) -1 )

− 1.1 to

− 0.7

− 1.7 to

− 1.0 X Weak Positive control (19-norethindrone) b fitted curve parameters Log e residual SD` NA 0.91-1.59 Top (% binding) NA 49-100 Bottom (% binding) NA

− 9 to 0 Hill Slope (log 10 (M) -1 ) NA

− 2.5 to

− 0.9 Solvent concentration DMSO ≤ 10% 4% X Negative control (octyltriethoxysilane) does not displace more than 25% of [ 3 H]-17 β -estradiol from the ER on average across all concentrations ≤ 25% <4% X

a Data were obtained from pages 25, 27 and 29 of the study report. b The EPA Guideline does not define a set of tolerance limits for 19-norethindrone. Acceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-nor ethindrone to be an accepta ble weak positive control. NA Not applicable



### The performance criteria for the competitive binding assay were ge nerally met with the exception two parameters for the reference chemical 17 β -estradiol: The bottom plateau (5%) for the third run exceeded the recommended range (

− 4 to 1%) and the Hill slope fell outside the recommended range (

− 1.1 to

− 0.7) for runs 2 and 3 (

− 1.4 and

− 1.7, respectively).

EPTC was tested over a concentr ation range that fully defined the top of the curve. The maximum percent binding 97.3-108.2% was within 25 percentage points of the lowest concentration of the estradiol standard 95.1-107. 4%. Examination across the runs indicated that the Hill slope for 17 β -estradiol decreased with each run and the Hill slope for 19- norethindrone was much higher in the second ru n than the first or third. Placements along the X-axis, and top and bottom platea us were consistent across runs.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: In the first independent run the mean specific binding was >96% at every solubl e concentration tested to EPTC. In the second independent run the mean specific binding was <101% at every soluble concentration tested. In the third independent run the mean specific binding for EPTC at 10

− 3 

### M was 69.6% of control.

Since EPTC was classified as "not interac tive" for the first two independent runs and "equivocal" in the third run, EPTC has a final classifica tion of not interactive.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 17 EPTC/ 041401 OCSPP 890.1250/ OECD None



### B. AGENCY COMMENTS: In the saturation binding experi ment, the protein concentrations used varied greatly between each run, and were approximately 3- to 6-fold greater than recommended. The mean K d 

### for [ 3 

### H]-17 β -estradiol was 0.331 nM and the estimated B max 

### was 74.55 fmol/100 μg protein for the prep ared rat uterine cytosol. The K d 

### for each run was within the expected Guidelin e range of 0.03 to 1.5 nM.

For the competitive binding experiment, EPTC reduced the specific binding only at the maximum concentration tested (10

− 3 

### M) in Runs 1 (96.2%) and Run 3 (69.6%). The estimated mean IC 50 

### was not calculated for EPTC as it did not achieve 50% binding. The mean IC 50 

### for the positive control was 3.2 × 10

− 6 

### M. The mean RBA was 0.029% for the weak positive control.

In two assay runs, EPTC reduced the specific bi nding of the radiolabeled estradiol at the highest concentration tested (10

− 3 

### M) from 95-107% in Runs 1 and 2 and in Run 3 the percent binding was re duced to 69.6% at 10

− 3 

### M. EPTC was classified as not interactive in in the first two runs and equivocal in the thir d run. Overall, EPTC is classified as not interactive in this assay.

C. STUDY DEFICIENCIES: The following deficiencies were not ed that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The weak positive control, 19-norethindrone, sh owed a top plateau of 49% binding in the second assay. The Reviewer noted that the weak positive control data were well outside the guidelines for acceptance, however, in the same run the positive and negative controls were within the accepta ble criteria.

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-13-2012 (MRID 48616405)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Michelle Sharpe-Kass, M.S. Date: 1/6/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 1/18/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 1/23/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 1/23/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).EPTC/ 041401 Estrogen Receptor Transcriptional Activation (2011) I Page I of 10 OCSPP 890.1300/ OECD 455


### Primary Reviewer:

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# fJJ!?];?> Secondary Reviewer: Minerva Mercado-Feliciano, Ph.D. DABT

### Signature:**~~** Health Effects Division Date: e-- 

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- Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903); OCSPP 890.1300; OECD 455.

PC CODE: 041401 DP BARCODE: D397177

TXR#: 0051762 CAS No.: 759-94-4

TEST MATERIAL (PURITY): EPTC 99.9% a.i.

SYNONYMS: S-ethyl N,N-dipropylcarbamothioate

CIT A TI ON: Willoughby, J 

### .A. (2011 ). EPTC: Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903)). CeeTox, Kalamazoo, MI. Laboratory report number 9142V-100349ERTA
, Nov 10, 2011. MRID 48616405. Unpublished.

SPONSOR: Gowan Company
, 370 S. Main Street, Yuma, AZ

TEST ORDER#: EDSP-041401-64

EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation (ERTA) assay (MRID 48616405), hERa-HeLa-9903 cells cultured in vitro 

### were exposed to EPTC (99.9% a.i

. , Batch # Pl 105096) at logarithmically increasing concentrations from 10

- 10 M to 10

- 3 

### Min DMSO (0.1 %) for 24 hours. The experiments were performed using 96-well plates and each EPTC concentration was tested in 6 replicates / plate. Cells were exposed to the test agents for 24

± 2 hr 

### to induce reporter (luciferase) gene products. Luciferase expression in response to activation of the estrogen receptor was measured using a proprietary method.

EPTC was tested up to the guideline maximum concentration of 10

- 3 

### M. No cytotoxicity was observed at any concentration of EPTC tested. The mean RPCMax for EPTC was 4.1 % for the first assay and 3.6% for the second assay, and the associated PCMa x was 10

- 5 

### and 10

- 6 

### M, respectively

. The reference chemicals**17~-estradiol** 

### and 17a-estradiol all performed within the acceptable range. However, cytotoxicity was observed at the highest concentrations of corticosterone (10

- 4 

### M) and 17-a methyl testosterone (10

- 5 

### M) in both assays. 17amethyltestosterone did not perform as expected
, having RPCMax of 13.7% in the first assay and 6.2% for the second assay ; Log PCso and Log PC10 could not be calculated.

In the main assays , the responsiveness of the cells to the very weak positive control 17amethyltestosterone was lower than the expected va lues
, indicating a decreased sensitivity of the assay to very weak agonists

. Although the conditions of this assay were not optimal to detectEstrogen Receptor Transc riptional Activation (2011) / Page 2 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455


### very weak activity, EPTC responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 6.2-13.7% PC. EPTC was only able to reach a maximum of 3.6-4.1% PC when tested up to the highest concentration possi ble based on cytotoxicity. Because the RPC Max 

### < PC 10 

### in both assay runs, EPTC was considered negative for estrogen receptor transcriptional activation in this test system.

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

COMPLIANCE: Signed and dated GLP, Quality Assurance and Data Confidentiality statements were provided.Estrogen Receptor Transc riptional Activation (2011) / Page 3 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: EPTC ( S -ethyl N,N -dipropylcarbamothioate) Description: Liquid, mw 189.3 g/mol Source: Gowan Company Lot/Batch #: P1105096, Exp. Feb 4 2015 Purity: 99.9 % w/w Solubility: Not reported (NR) Volatility: NR Stability: NR Storage conditions: Ambient Vapor pressure: NA CAS #: 759-94-4 Structure:

N S O CH

3

CH 3 CH 3



### 2. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: NR Catalogue and Batch #: NR Purity: NR CAS # : 50-28-2



### 17 α -estradiol (weak estrogen) Supplier: NR Catalogue and Batch #: NR Purity: NR CAS # : 57-91-0 

### Corticosterone (negative compound) Supplier: NR Catalogue and Batch #: NR Purity: NR CAS # : 50-22-6



### 17 α -methyltestosterone (very weak agonist) Supplier: NR Catalogue and Batch #: NR Purity: NR CAS # : 58-18-4 

### 3. Vehicle(s) Solvent: DMSO, Supplier and lot not reported Solvent control (final concentration): 0.1%



### B. METHODS

1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Bioresour ces Cell Bank and were verified to be free of mycoplasma infection using a DNA fluorochrome procedure. Cells were maintained in Eagles Minimum Essential Medium without phenol red, supplem ented with kanamycin (60 mg/L) and 10% dextran-coated charcoal-treat ed fetal bovine serum (source not reported), in an incubatorEstrogen Receptor Transc riptional Activation (2011) / Page 4 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455



### under 5% CO 2 

### at 37oC. Upon reaching 75-90% confluen ce, cells were subcultured prior to exposure to the test material. Cells used in the range finding study were passage 21. New cell vials were brought up from cryopreservation and maintained for the two valid runs (8/23/2011 and 8/25/2011) and we re passages 19 and 20 prior to seeding into plates.

2. Transcriptional Activation Assays: For each test, cells were plated at a density of approximately 1 × 10 4 

### cells/100 μL medium/well in a 96 well plate and allowed to attach for at least 3 hours. The growth media was replac ed with media containing serial log dilutions of EPTC in DMSO. Cells were incubated for 24 ± 2 hours at 37±1°C. The total final concentration of DMSO was 0.1%. Cytotoxic ity was determined by propidium iodide (PI) uptake, with 125 μ M digitonin as a positive c ontrol for cytotoxicity.

It was stated that the transcriptional activati on of the estrogen receptor was determined as described in CeeTox Standard Operating Pr otocol (SOP) 2041. A list of reagents was provided, but the assay reagent was classified as proprietary information, and the SOP was not provided.

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 10

− 3 

### to 10 − 6.5 

### M was conducted to determine the appropri ate concentration range and to determine concentrations resulting in insolubility and/or cytotoxicity. Solubility was determined by visual inspection of the test chemicals and cont rols final concentrations in culture medium.

b. Proficiency Chemicals: The responsiveness of the test system was tested on March 5, April 12, and April 28, 2011 (MRID 48843501), using cells at Passage 15, 25 and 28, respectively. Based on the assay dates and cell passage numbers, it is unlikely that the cells used for proficiency testing are from the same frozen stock as the cells used in the main assay. The cells were tested using the follo wing set of proficiency chemicals, and each chemical was tested in duplicate on separate days:

Compound CAS No. Concentration Range (M) Expected Response a Notes Diethylstilbestrol (DES) 56-53-1 10

− 14 to 10

− 8 Positive --- 17 α -Ethynyl estradiol (EE) 57-63-6 10

− 14 to 10

− 8 Positive --- Hexestrol 84-16-2 10

− 13 to 10

− 7 Positive --- Genistein 446-72-0 10

− 12 to 10

− 5 Positive Cytotoxic at 0.01 b , 0.1, and 1 mM Estrone 53-16-7 10

− 12 to 10

− 6 Positive --- Butyl paraben 94-26-8 10

− 11 to 10

− 4 Positive Cytotoxic at 0.1 b and 1 mM 1,3,5-Tris(4-hydroxyphenyl)benzene c 15797-52-1 10

− 12 to 10

− 5 Positive Cytotoxic at 100μM. PC max approx. 50% of PC. Binds to hER α and has ER antagonistic activity Dibutyl phthalate (DBP) 84-74-2 10

− 11 to 10

− 4 Negative d Cytotoxic at 1 mM Atrazine 1912-24-9 10

− 11 to 10

− 4 Negative Cytotoxic at 1 mM b Corticosterone 50-22-6 10

− 10 to 10

− 4 Negative If not cytotoxic at 1 mM, then that should be the highest tested concentration a Positive = RPC max ≥ 10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC max fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs b Cytotoxicity is expected to be close to 80% at this concentration. c Compound selected to challenge solubility and cytotoxicity. d DBP is negative for ER α mediated transcriptional activation, but may not be negative for non-ER β mediated transcriptional activation. A positive result would indicate that the sy stem is detecting activity other than that due to pure ER α , and is therefore unacceptable.Estrogen Receptor Transc riptional Activation (2011) / Page 5 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455



### c. Reference Chemicals: To ensure the stability of th e response from the cell line, six concentrations of each of the following reference chemicals were included on separate plates in the current assay:

Reference Chemical CAS No. Concentration Range Class 17 β -estradiol 50-28-2 10

− 14 to 10

− 8 Strong estrogen 17 α -estradiol 57-91-0 10

− 12 to 10

− 6 Weak estrogen Corticosterone 50-22-6 10

− 10 to 10

− 4 Negative compound 17 α -methyltestosterone 58-18-4 10

− 11 to 10

− 5 Very weak agonist



### 3. Data analysis: To obtain the relative transcriptional activity to the 1 nM E2 positive control (PC), the luminescence signals from the concurrent plate were analyzed by subtracting the mean value of the vehicle control from each well value to normalize the data; each normalized value was then divided by the mean value of the normalized PC. The resulting value was multiplied by 100 in order to express relative transcriptional activity as a percentage of the PC. The maximum res ponse relative to the positive control (RPC max 

### ) for EPTC was calculated, but the software used wa s not reported. The test material was defined as negative for inducing es trogen receptor transcriptio nal activation if the RPC Max 

### < PC 10 

### in at least 2 of 2 (or 2 of 3) runs. LogEC 50 

### and Hill slope values are calculated only if a positive response is observed. Coefficients of variation (CV) were calculated for the luminescence data triplicates. Concentrations showing >20% cytotoxi city or evidence of insolubility were excl uded from analyses.

4. Definitions

EC 50 

### = concentration of agonist that induces a response ha lfway between the baseline (bottom) and maximum (top) response

PC 10 

### = concentration of a test chemical at which the response is 10% of the response induced by the positive control (E2 at 1 nM) in each plate

PC 50 

### = concentration of a test chemical at which the response is 50% of the response induced by the positive control (E2 at 1 nM) in each plate

RPC Max 

### = maximum level of response induced by a test chemical, expressed as a percentage of the response induced by the positive control (1 nM E2) on the same plate

PC Max 

### = concentration of a test chemical inducing the RPC Max



### II. RESULTS

A. PRELIMINARY TEST: In order to identify a suitabl e top concentration for use in the transcriptional activation assays, preliminary cytotoxicity and precipitation assays were conducted. Precipitation was not obs erved in any of the EPTC so lutions. The results of the cytotoxicity test are presented in Table 1. No cytotoxicity was observed for EPTA at any concentration tested. Based on thes e results, concentrations of 10

− 10 

### M to 10 − 3 

### M were selected for the assay.Estrogen Receptor Transc riptional Activation (2011) / Page 6 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455

TABLE 1. Preliminary Test for Solubility, Cyto toxicity, and Concentration-Selection for EPTC a Concentration (M) % Viability b Comments
10

− 3 82

No precipitation observed for any concentration 10

− 3.5 87 10

− 4 91 10

− 4.5 92 10

− 5 97 10

− 5.5 97 10

− 6 97 10

− 6.5 99 E2 1nM NR VC b NR a Data were obtained from page 18 of the study report. b VC = Vehicle control


### B. Positive and Negative Reference Chemicals

1. Proficiency Chemicals: The laboratory proficiency assa ys using the required reference compounds were not included in th e original study report, but were provided to the Agency at a later date (MRID 48843501). The responsiven ess of cells to the required proficiency chemicals was performed in duplicate on different days for each chemical. The reported responses are in Table 2.

TABLE 2. Proficiency Chemicals
Compound Expected Response Lab Response Run 1 Run 2 Run 3 Diethylstilbestrol Positive Positive Positive NA 17 α -Ethynyl estradiol Pos itive Positive Positive NA Hexestrol Positive Positive Positive NA Genistein Positive Positive Positive NA Estrone Positive Positive Positive NA Butyl paraben Positive Positive Positive NA 1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive NA Positive Positive Dibutyl phthalate Negativ e Negative Negative NA Atrazine Negative Ne gative NA Negative Corticosterone Negative Negative NA Negative NA = not applicable. The chemical was not tested at this time.



### 2. Reference Chemicals: Values derived from the concentration response curve ( e.g ., Log PC 50, 

### Log PC 10, 

### Log EC 50, 

### Hill slope) for the four concurrently run reference materials are included in Table 3. The reference chemicals 17 β -estradiol and 17 α -estradiol all performed within the acceptable range. However, cy totoxicity was observed at the highest concentrations of corticosterone (10

− 4 

### M) and 17- α methyltestosterone (10

− 5 

### M) in both assays. The Log PC 50 

### and Log PC 10 

### for 17 α -methyltestosterone were not calculated. Visual inspection of the raw data indicated that 17 α -methyltestosterone had an RPC Max 

### of 13.7% in the first assay and 6.2% for the second assay. The PC 10 

### for 17 α -methyltestosterone was not calculated, and no reason was given for not ca lculating it despite reaching >10% PC. The Reviewers assume it was because higher c oncentrations did not result in increased transcriptional activation, as woul d be expected. Based on this, a PC 10 

### and PC 50 

### could not be calculated for 17 α -methyltestosterone. It was stated th at these deviations from the ranges suggested in the OCSPP guideline were mi nor and not considered to impact the interpretation of results.Estrogen Receptor Transc riptional Activation (2011) / Page 7 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455

TABLE 3. Performance Criteria for Reference Chemicals a

Reference Chemical Parameter Acceptable Range Values Acceptable Run 1 Run 2 Yes No 17 β -estradiol Log PC 50 − 11.4 to

− 10.1

− 10.4

− 10.2 X Log PC 10 <

− 11

− 11.3

− 11.3 X Log EC 50 − 11.3 to

− 10.1

− 10.3

− 10.2 X Hill Slope 0.7 to 1.5 1.1 1.1 X Test range 10

− 14 to 10

− 8 M 10

− 15 to 10

− 8 M 10

− 15 to 10

− 8 M X 17 α -estradiol Log PC 50 − 9.6 to

− 8.1

− 8.4

− 8.6 X Log PC 10 − 10.7 to

− 9.3

− 9.5

− 9.6 X Log EC 50 − 9.6 to

− 8.4

− 8.4

− 8.6 X Hill Slope 0.9 to 2.0 1.2 1.5 X Test range 10

− 12 to 10

− 6 M 10

− 13 to 10

− 6 M 10

− 13 to 10

− 6 M X Corticosterone Test range 10

− 10 to 10

− 4 M 10

− 11 to 10

− 4 M 10

− 11 to 10

− 4 M X 17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1 NC NC X Log PC 10 − 8.0 to

− 6.2 NC NC X Test range 10

− 11 to 10

− 5 M 10

− 12 to 10

− 5 M 10

− 12 to 10

− 5 M X a Data were obtained from page 21 of the study report. NC = not calculable.



### C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Data for the vehicle and pos itive controls are included in Table 4. The overall mean TA value, expresse d in arbitrary light units, for the vehicle control was 9779 for the first assay and 11742 for the second assay. The mean value for the positive control was 205092 for the first a ssay and 187875 for the second assay. The PC 50 

### (50% of the maximum response) for E2 in th is assay is 107435 for the first assay and 99808 for the second assay and the PC 10 

### (10% of the maximum response) is 29310 for the first assay and 29355 for the second assay.

TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a Sample Vehicle Control Positive Control b

Runs Mean SD Mean SD Fold Induction c

1 9779 1490 205092 40377 21 2 11742 1469 187875 66332 16 a Data were obtained from pages 29-30 of the study report. b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC)



### 2. Test Material: Relative (to the PC) transcriptional ac tivation at each concentration of the test chemical during the two assay runs is pr esented in Table 5. Th e concentration-response curves depicting fold induction of relative tr anscriptional activation are presented in Figure 1 below. The mean RPC Max 

### was 4.1% at 10
− 5 

### M for the first assay and 3.6% at 10

− 6 

### M for the second assay. However, the lack of responsiveness of th e assay to the weak agonist 17 α -methyltestosterone precluded the detection of a weak agonist.Estrogen Receptor Transc riptional Activation (2011) / Page 8 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455

TABLE 5. Relative Transcriptional Activation (RTA) of EPTC a
Parameter RTA (mean ± SD); % of Positive Control (PC) Run 1 Run 2 Conc. (M) Mean SD Mean SD 10

− 3 3.0 1.2 2.6 1.2 10

− 4 3.3 1.2 3.3 1.5 10

− 5 4.1 1.2 3.4 0.4 10

− 6 3.8 1.3 3.6 1.5 10

− 7 0.5 1.5 1.4 0.7 10

− 8 1.4 1.4 1.4 1.7 10

− 9 0.4 1.4 0.7 1.6 10

− 10 0.4 0.9 1.0 1.3 Log EC 50 b NA NA Hill Slope b NA NA RPC Max 4.1% 3.6% PC Max 10

− 5 10

− 6
PC 50 NA NA PC 10 NA NA a Data were obtained from pages 19 and 20 of the study report. NA= Not ApplicableEstrogen Receptor Transcriptional Activation (2011) / Page 9 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455

FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of EPTC.



### 3. Performance Criteria: The laboratory proficiency assays using the required reference compounds were not included in the study report and were not available to the reviewer. 17 β -estradiol and 17 α -estradiol all performed within the acceptable range. However, cytotoxicity was observed at the highest concentrations of corticosterone (10

− 4 

### M) andEstrogen Receptor Transc riptional Activation (2011) / Page 10 of 10 EPTC/ 041401 OCSPP 890.1300/ OECD 455



### 17- α methyltestosterone (10

− 5 

### M) in both assays. The Log PC 50 

### and Log PC 10 

### for 17 α -methyltestosterone was not calcu lated. Visual inspection of the raw data indicated that 17 α -methyltestosterone had an RPC Max 

### of 13.7% in the first assay and 6.2% for the second assay. In the first assay, 17 α -methyltestosterone induced tran scriptional activation greater than 10% PC at 10 -8 

### M, which is within the range of acceptable PC 10 

### values; however higher concentrations did not result in further incr eased activity. The second assay shows a dosedependent increase in luci ferase activity from 17 α -methyltestosterone, however while maximum induction does reach the acceptable range of PC 10 

### values it does not reach the acceptable range of PC 50 

### values. Because of this, proper PC 10 

### and PC 50 

### values could not be calculated for 17 α -methyltestosterone. Triplicate samples of the PC and the VC were included in each plate, and the mean luciferase activity of the PC was greater than 4-fold that of the mean vehicle control on each pl ate. Based on calculations performed by the Reviewers, the fold-induction corresponding to PC10 of concurre nt PC was greater than 1+2 standard deviations of the fold-induction value of the concurrent VC, as expected. There was some inter- and intra-assay variability, w ith some instances of the CV being greater than 20%, indicating an unrel iable calculation of the PC 10



### .

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration of EPTC for use in the transcriptional activation assays was 10

− 3 

### M. Cytotoxicity (>20% reduction in cell viability) was not observed at any test concen tration of EPTC. In two valid independent runs of the transcriptional activation assay, EPTC did not result in an increase in luciferase activity at any of the viable concentrations tested (RPC max 

### <10%). The highest mean induction value was 4.1% compared to vehicle c ontrol. EPTC was tested across a soluble concentration range (10

− 10 

### to 10 − 3 

### M) and was determined to not be an agonist of human estrogen receptor alpha (hERa) using the HeLa-9903 cell line.

B. AGENCY COMMENTS: EPTC was tested up to the guideline maximum, 10

− 3 

### M. No cytotoxicity was observed at any concentr ation of EPTC tested. The mean RPC Max 

### was 4.1% at 10
− 5 

### M for the first assay and 3.6% at 10

− 6 

### M for the second assay. Although the conditions of this assay were not optimal to de tect very weak activity, EPTC responses were similar to those of the negative control cortic osterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 6.2-13.7% PC. EPTC was only able to reach a maximum of 3.6- 4.1% PC when tested up to the highest concentration possible based on cy totoxicity. Because the RPC Max 

### < PC 10 

### in both assay runs, EPTC was considered negative for estroge n receptor transcriptional activation in this test system.

C. STUDY DEFICIENCIES: The following deficiencies were noted:

 17 α -methyltestosterone did not induce transcriptional activat ion equal to 50% or 10% of the positive control in either run; therefore a PC 10 

### and PC 50 

### could not be calculated. This indicates weak responsiveness of th e assay to the very weak agonist.

 Cytotoxicity was observed in the reference chemicals 17 α -methyltestosterone and corticosterone; cytotoxicity should not occur in any of the recommended reference chemical concentrations.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 1 of 89 Data Requirement: EPA DP Barcode 402838

OECD Data Point 229

EPA MRID 48827602 EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: EPTC Purity: >99.9%

Common name Chemical name: IUPAC: S-ethyl dipropyl(thiocarbamate)

CAS name:

CAS No.: 759-94-4 Synonyms: Eptam® Technical.

EPA PC Code: 041401

Primary Reviewer: Joan Gaidos Signature: Senior Scientist, Cambridge Environmental, Inc. Date: 9/25/12

Secondary Reviewer: Teri S. Myers Signature: Senior Scientist, Cambridge Environmental, Inc Date: 9/27/12

Primary Reviewer: Frank T. Farruggia, Ph.D. Date: 3/22/13

EPA/OPP/EFED/ERB3

Secondary Reviewer(s): Amy Blankinship Date: 6/5/15

EPA/OPP/EFED/ERB3

Date Evaluation Completed: 6/5/15

CITATION: York, D.O. 2012. EPTC - Short-term Reproduction Assay with the Fathead Minnow ( Pimephales promelas ).
Performed by Smithers Viscient, Wareham, Massachusetts, Laboratory Project No. 12791.6156. Submitted by Gowan
Company, Yuma, Arizona. Completion date May 4, 2012

. 

# FRANK FARRUGGIA Digitally signed by FRANK FARRUGGIA DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=FRANK FARRUGGIA, dnQualifier=0000048865 Date: 2015.06.11 08:16:39 -04'00'



## AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.15 17:34:16 -04'00'

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 2 of 89 Note: The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of
each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

Guideline recommendations are provided in italics; these recommendations should remain visible in the
completed DER.

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to aid in
preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test
Guidelines, nor to provide any guidance on how the study should be conducted.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 3 of 89 EXECUTIVE SUMMARY:

The 21-day short-term reproduction as say of EPTC with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish, 16 spawning groups (2 males and 4 females in each group; 6 months old) were exposed to EPTC (>99.9% purity) at nominal concentrations of 0 (control), 0.08, 0.80, and 8.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.014), 0.069, 0.69, and 6.9 mg
a.i./L. The test system was maintained at 24 to 25 o C and a pH of 6.7 to 7.4.

Mean survival was 100, 100, 75, and 13% for males and 94, 100, 38 and 6% for females in the control, low, mid, and high treatment groups, respectively; overall survival (both sexes) was 96, 100, 50, and 9%
respectively. Due to the high mortality at the high co ncentration, these data were not used for statistical analysis. Mortality was significantly different in the mid and high treatment groups compared to the negative control (p<0.05; Fisher’s Exact Test). There were no no table observations with rega rds to behavior or changes in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad, in the control or treated groups. There were no notable observations of secondary sex characteristics or clinical signs for any treatment group compared to the controls. Male and female growth parameters (wet weight and total length)
were not significantly different from the control.

Spawning in each replicate occurred at least every 4 days and mean fecundity in controls was 22 eggs/female/day/replicate (range 18-25 E/F/D); fertiliz ation success in the control group averaged 96%. Fecundity was significantly (p<0.05; Dunnett’s) reduced by 75% at the mid treatment concentration.

There were no significant differences between controls and the low and mid treatment groups for male orfemale gonado-somatic index (GSI). Male tubercle scores was marginally significantly (p=0.057; MannWhitney) reduced compared to the control (26 vs 41 in control) at the 0.69 mg a.i./L treatment level and was approaching significance (p=0.086) in the 0.069 mg ai/L treatment level (32 vs. 41); none of the
surviving females were found to have tubercles.

There were no treatment-related histopathologic observations in males. In female fish, there was an increase in incidence of minimal to moderate oocyte atresia at the mid treatment level compared to the control; however, the smaller sample size (n=6) prevented further interpretation of this finding. There were no treatment-related
effects at any test level for gonadal staging. There were no other significant effects on other endpoints. For

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 4 of 89 male vitellogenin (VTG), the reported control values were unusually high (comparable to control female values)
even after re-analysis; the values in the other treatment groups were also comparable to female VTG values. No obvious reasons for these high values were reported and as such, these data are not considered reliable for interpretation. Sex steroids were not measured in this study.

The validity criteria were met except that dissolved oxygen fell slightly below the recommended 60% of saturation (58%) in one test chamber; the chambers were cleaned and the levels returned to above the
recommended value, and this deviation did not sufficiently impact the interpretation of the study.

In regards to male VTG, the inability to evaluate this endpoint is a major limitation in this study. However, it is noted that there were no effects on female VTG or male GSI or histopathology. This information along with the information provided in the remaining Tier 1 battery were considered collectively for evaluating potential effects in male fish from EPTC exposure. Based on this information, additional information regarding impacts
to male VTG is not requested.

As such, this assay satisfies the EDSP Tier 1 Test Order requirement for a Fish Short-Term Reproduction Assay (OCSPP Guideline 890.1350).

Results Synopsis

Test Organism age at test initiation: 6 months Mean body weight at test initiation (if measured): Male 2.9 g; Female 1.6 g Mean length at test initiation (if measured): Not reported. Test Type (Flow-through, Static, Static Renewal): Flow-throughData Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 5 of 89Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with EPTC.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFM4FMFMF0.069 No No YesNo No No No No NRLNo NA NA NA NA0.69YesNo NoNo No No No No NRLNo NA NA NA NA6.93NA NA NA NA NA NA NA NA NA NA NA NA NA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable,NRLNot reliable,.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.3Endpoints at the high treatment level were not statistically analyzed due to high mortality.4Due to unusually high control male VTG values, these data are not considered reliable for interpretation.

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Page 6 of 89 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 890.1350: “Fish Short-Term Reproductive Assay” and OECD 229 (2009). The following deviations were noted:

1. The unionized ammonia and residual chlorine in the test water were not reported. The OCSPP 890.1350 performance criteria establish maximum levels

for these values and it is unclear if the maximum recommendations were

exceeded.

2. The dissolved oxygen fell to 58% of saturation in the control replicate B on Day 10 of the exposure which is slightly below the recommended 60% of saturation.
The test chambers were cleaned and the levels were above 60% by the next
day.

These deviations do not have an impact on the acceptability of this study.

COMPLIANCE: Signed and dated No Data Confidentiality, GLP, and Quality Assurance
statements were provided. This study was conducted in compliance with all pertinent U.S. EPA Good Laboratory Practice regulations with the following exceptions: routine dilution water and food contaminant screening analyses were conducted following standard validated methods, this exception has no
impact on the study results.

A. TEST MATERIAL: EPTC; CAS# 759-94-4.

Description: Not reported.

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or
Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : P1105096

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 7 of 89 Purity: >99.9%

Impurities: None reported

Stability of Compound: Analytical verification of the test material from time 0, 6, 13, and 21 days yielded recoveries of 78.8% to 91.3% of nominal concentrations. Stock solution fortified with EPTC at concentrations of 0.04, 0.8, and 8.0 mg a.i./L yielded recoveries of 83.6 to 107%. There was no pattern of decline during the study period, indicating the test material was stable under the test

conditions.

Storage Conditions of Test Chemicals: Stored at room temperature in dark, ventilated cabinet.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 8 of 89B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: Fathead Minnow EPA recommends fathead minnow (Pimephales promelas).Species scientific name: Pimephales promelasSpecies strain (if stated): Not reportedWere fish obtained from asingle laboratory stock?Yes EPA recommends that fish be from a single laboratory stock.Were acclimation conditionssame as definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive

test.Acclimation period:13 days EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 9 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:1 mortality (<1%)Mortalities during the 7 days prior topre-exposure period were <1%.Fish did not receive any treatment fordisease during acclimation period.Behavioral abnormalities or clinical signswere not reported. EPA recommends that mortality during the 7 days prior to the pre-exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5-10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the fish not be used.Type of food:Frozen brine shrimp EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body

condition.Source of food:Not reportedRepresentative samples of the food sourcewere periodically analyzed for pesticides,PCBs, and toxic metals by GeoLabs, Inc.and analyte levels were below LOC specifiedby ASTM (2005).Frequency of feeding:2 times/dayDetails on feeding:None.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 10 of 89Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:6 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months

old.Mean weight of males at testinitiation (if determined):2.9 g Based on 20 males used to stockaquaria for pre-exposure phase. EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each sex.Range of individual weights(males) at test initiation (ifdetermined):2.4 to 3.4 Individual weights within ± 20% of theestimated mean.Mean weight of females at testinitiation (if determined):1.6 g Based on 20 females used to stockaquaria for pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):1.3 to 2.0 Individual weights within ± 26% of theestimated mean.Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedData Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 11 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsDuration of pre-exposure phase: 13 days EPA recommends a minimum of 14 days.Were pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:36Extra replicates were established toaccount for a potential lack of spawningin some chambers and/or mortalityduring this phase. EPA recommends that additional tanks set up at the beginning of pre-exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2Number of females per tank:4Pre-exposure fecundity:>15 eggs/female/reproductive day/replicate EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 12 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of spawns during preexposure:>two times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:None.C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this assay.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 13 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem:Intermittent flow proportionaldiluters Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate: Ca. 45 mL/minute or 2.7L/hr and 6.6 volumereplacements/day.Calculated by the reviewer based on 0.5L/cycle, 132 cycles/24 hrs and 60minutes/hr. Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.Details on toxicant mixing forflow-through systems:Flow-splitting chambers were usedbetween diluter cells and the 4 replicatetest vessels.Treatment recoveries were not measuredprior to test initiation to indicate if diluterhad reached equilibrium.Flow splitting accuracy not reported.Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within 10%.Aeration?Not reported EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60%
saturation).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 14 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water: Well water EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?YesTest vessel type/materials: Glass aquaria withsilicone sealant andnylon.EPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:20 cm wide x 39 cmlong x 25 cm high;solution depth 13 cm. EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 15 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsFill volume:10 L EPA recommends 10 L solution per tank.Spawning substrate material: 4-inch diameter agedPVC cut in half (arches)and into 7 cm sections. EPA recommends that each tank contain three semi-circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split lengthwise.Spawning substrate size:PVC cut in half (arches)and into 7 cm sections.Each substrate placed on screened glasscollection tray (13 x 11 x 3 cm, L x W xH), covered with 0.5 cm2stainless steelmesh.Additional details on exposuresystem:None.Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)242524.51Continuously EPA recommends temperature 25±1 o C; inter-replicate and inter-treatment differentials should not exceed 1 o C.pH 6.77.47.11Daily EPA recommends pH 6.5 to 9.0.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 16 of 89ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsDissolved oxygen (mg/L)4.88.16.51Daily EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L asCaCO3)20 26 231Weekly EPA recommends total alkalinity >20 mg/L as CaCO 3.Hardness (mg/L as CaCO3) 60 72 661WeeklyTotal organic carbon (mg/L) 0.11 0.160.14Monthly EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (Pg/L) Not reported Not reported Not reportedNot reported EPA recommends that unionized ammonia in the dilution water be <1

P g/L.Residual chlorine (Pg/L) Not reported Not reported Not reportedNot reported EPA recommends that residual chlorine in dilution water be <10 P g/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 17 of 89ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsConductivity (Pmhos/cm) 330 450 3901Weekly General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at the highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.Abbreviations:NANot applicable.1Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 18 of 89D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes EPA recommends conducti ng a range-finder
if 96-hour LC 50 data for the fathead minnow are unavailable.If yes, what was the method fordetermining the highest testconcentration in the rangefinder?0 (control), 2.5, 5.0, 10,20, and 40 mg a.i./L EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is appropriate.Species: Pimephales promelasLife stage:Not reported EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the

test.Test duration:96 hours EPA recommends a 96-hour exposure.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 19 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:At test termination,17% and 100%mortality was observedin the 20 and 40 mga.i./L treatment level. EPA recommends conducti ng a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the derived 96-hr LC 50, may be selected as the highest exposure concentration in the 21-

day test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 20 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in thedefinitive test:The acute toxicity 96-hr LC50value with fathead minnowswas reported determined to be25 mg a.i./L (95% CI of 20 to31 mg a.i./L).Nominal concentrations selectedbased on range finding studyresults and consultation withsponsor. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):Not reportedSeparation of testconcentrations:0 (control), 0.8, 0.80, and 80mg a.i./L EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or ten-fold) is scientifically
acceptable 1 .Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 21 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsAre nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean measuredLimit of quantification (LOQ):<0.014 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reported.Frequency of measurement: 0, 6, 13, and 21 days It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 22 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsWas the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre-exposure. Tanks are then ranked according to preexposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of male fish perreplicate at test initiation:2 EPA recommends 2 males per replicate.Number of female fish perreplicate at test initiation:4 EPA recommends 4 females per replicate.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 23 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsWas a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed 0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8 (light:dark).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing andAssessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 24 of 89ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’ssurface:560-980 luxLight was provided by SylvaniaOktron®fluorescent bulbs andintensity was measured with aVWR light meter. EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:All exposure solutions werereported as clear and colorlesswith no undissolved testsubstance observed.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 25 of 89Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with EPTC.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)0 <LOQ NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Treatment 10.080.0691.7Treatment 20.80.696.2Treatment 38.06.92.1Diluter StockNANANAAbbreviations:CVCoefficient of variation.LOQ=0.014 mg a.i./L.E. ObservationsBiological Endpoints: Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination(Day 21),secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal stagingand histopathology, and plasma VTG were evaluated.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 26 of 89Were raw (individual) data provided?Yes EPA recommends that observations of survival, fecundity, fertiliz ation success, secondary sex characteristics, and other clinic al signs occur at least daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, and plasma sex steroids (testosterone and 17ȕ -estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal beha vior.I.RESULTS AND DISCUSSIONFor all endpoints, the high treatment level was not statistically analyzed due to high mortality.A.ResultsMean male survival values were 100, 100, 75, and 13% and female survival values were 94, 100, 38 and 6% in the mean-measured <LOQ(control), 0.069, 0.69, and 6.9 mg a.i./L treatment levels, respectively (Table 9). Overall survival (both sexes) was 96, 100,50, and 9% in themean-measured <LOQ (control), 0.069, 0.69, and 6.9 mg a.i./L treatment levels, respectively.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 27 of 89Table 9: Adult Fish Survival in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]MalesFemalesN1# Surviving% SurvivalN1# Surviving% SurvivalControl (<LOQ)881001615940.069 8 8 100 16 16 1000.69 8 6 75 16 6 386.928 2 13 16 1 6Abbreviations:NANot applicable.1Total number of fish at test initiation.2Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 28 of 89Mean male body weight values were 3.73, 3.53, and 3.52 g and mean female body weight values were 1.39, 1.43 and 1.64 g in the meanmeasured <LOQ (control), 0.069, and 0.69 mg a.i./L treatment levels, respectively (Table 10). Mean male body length values were 65.7, 64.0and 64.4 mm and female body length values were 50.7, 51.6 and 52.0 mm in the mean-measured <LOQ (control), 0.069, and 0.69 mg a.i./Ltreatment levels, respectively.Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDControl (<LOQ) 4 3.73 0.18 4 1.39 0.17 4 65.7 1.4 4 50.7 1.10.069 4 3.53 0.61 4 1.43 0.11 4 64.0 4.8 4 51.6 0.660.69 4 3.52 0.22 4 1.64 0.11 4 64.4 2.8 4 52.0 0.516.914 NA NA 4 NA NA 4 NA NA 4 NA NAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.1Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.

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Page 29 of 89Mean fecundity values were 22, 27 and 5.6 and fertilization success was 96, 96, and 85 in the meanmeasured <LOQ (control), 0.069, and 0.69 mg a.i./L treatment levels, respectively (Table 11).

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)
[mean-measured] Fecundity 1 Fertilization Success (%) 2

Mean ±SD Mean ±SD

Control (<LOQ) 22 3.0 96 1.4

0.069 27 5.0 96 1.8

0.69 5.6 3.1 85 20

6.9 3 NA NA

Abbreviations: NA Not applicable. ND Not determined.

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2 Fertilization success (%) is calculated as the number of embryos divided by the number of eggs,
multiplied by 100.

3 Endpoints at the high treatment level were not statistically analyzed due to high mortality.
LOQ=0.014 mg a.i./L.

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Page 30 of 89 Median male tubercle scores were 41, 32 and 26 in the mean-measured <LOQ (control), 0.069, and 0.69 mg a.i./L treatment levels, respectively (Table 12). None of the surviving females were found to

have tubercles.

Table 12: Nuptial Tubercle Score in Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)
[mean-measured] Males Females

n Median

Tubercle Score n Median

Tubercle Score

Control (<LOQ) 4 41 4 0

0.069 4 32 4 0

0.69 4 26 4 0

6.9 1 4 NA 4 NA

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

1 Endpoints at the high treatme nt level were not statistically analyzed due to high mortality.
LOQ=0.014 mg a.i./L.

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Page 31 of 89Mean male GSI was 1.5, 1.2 and 1.9 and mean female GSI was 13, 12 and 16 in the meanmeasured <LOQ (control), 0.069, and 0.69 mg a.i./L treatment levels, respectively (Table 13).

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)
[mean-measured] Males Females

n Mean GSI 1
(%) ±SD n Mean GSI 1
(%) ±SD

Control (<LOQ) 4 1.5 0.32 4 13 0.80

0.069 4 1.2 0.29 4 12 2.2

0.69 4 1.9 0.17 4 16 1.7

6.9 2 4 NA NA 4 NA NA

Abbreviations: NA Not applicable.

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x

100.

2 Endpoints at the high treatment level were not statistically analyzed due to high mortality.
LOQ=0.014 mg a.i./L.

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Page 32 of 89 Median male and female gonadal stages were 2, 2 and 3 in the mean-measured <LOQ (control), 0.069,and 0.69 mg a.i./L treatment levels, respectively (Table 14). The study authors did not indicate treatmentrelated affects at any test level for gonadal staging.

Table 14: Gonadal Staging in Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)
[mean-measured] Males Females

n Median Stage 1 n Median Stage 2

Control (<LOQ) 8 2 15 2

0.069 8 2 16 2

0.69 6 3 6 3

6.9 3 NA NA NA NA

Abbreviations: J Juvenile. NA Not applicable. ND Not determined. UTS Unable to stage.

1 The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped, 1=early
spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2 The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early
development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory.

3 The one male and one female were not examined by the pathologist. LOQ=0.014 mg a.i./L.

In male fish, there was one incidence of mild testicular degeneration at the 0.069 mg a.i./L group. Otherwise, there were no treatment-related histological effe cts or differences in gonadal stages in testes in any of the treatment groups (Table 15). In female fish, there was an increase in incidence of minimal to moderate oocyte atresia at the 0.69 mg a.i./L treatment level compared to the control (Table 17). However, the smaller sample size (n=6) prevented further interpretation of this finding.

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Page 33 of 89 Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased Proportion of Spermatogonia Presence of

Testis-Ova Increased

Testicular
Degeneration Duct

Mineralization Interstitial cell (Leydig)
hypertrophy/hype
rplasia

n Incidence n Incidence n Incidence n Incidence n Incidence

Control (<LOQ) 0 8 8 8 8 8 8 NA NA 8 8

1 8 0 8 0 8 0 NA NA 8 0

2 8 0 8 0 8 0 NA NA 8 0

3 8 0 8 0 8 0 NA NA 8 0

4 8 0 8 0 8 0 NA NA 8 0

0.069 0 8 8 8 8 8 7 NA NA 8 8

1 8 0 8 0 8 0 NA NA 8 0

2 8 0 8 0 8 1 NA NA 8 0

3 8 0 8 0 8 0 NA NA 8 0

4 8 0 8 0 8 0 NA NA 8 0

0.69 0 6 6 6 6 6 6 NA NA 6 6

1 6 0 6 0 6 0 NA NA 6 0

2 6 0 6 0 6 0 NA NA 6 0

3 6 0 6 0 6 0 NA NA 6 0

4 6 0 6 0 6 0 NA NA 6 0

6.9 0 NA NA NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA NA NA

2 NA NA NA NA NA NA NA NA NA NA

3 NA NA NA NA NA NA NA NA NA NA

4 NA NA NA NA NA NA NA NA NA NA

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.
LOQ=0.014 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 34 of 89Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl (<LOQ) 0 8 8 8 8 8 8 8 8 8 81 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 00.0690 8 8 8 8 8 8 8 8 8 81 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 0Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 35 of 89Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.690 6 6 6 6 6 6 6 6 6 61 6 0 6 0 6 0 6 0 6 02 6 0 6 0 6 0 6 0 6 03 6 0 6 0 6 0 6 0 6 04 6 0 6 0 6 0 6 0 6 06.920 NA NA NA NA NA NA NA NA NA NA1 NA NA NA NA NA NA NA NA NA NA2 NA NA NA NA NA NA NA NA NA NA3 NA NA NA NA NA NA NA NA NA NA4 NA NA NA NA NA NA NA NA NA NA1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.2Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.

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Page 36 of 89 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell
Hyperplasia/
Hypertrophy Decreased Yolk

Formation Aggregates of
macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

Control (<LOQ) 0 14 12 14 14 14 14 NA NA

1 14 2 14 0 14 0 NA NA

2 14 0 14 0 14 0 NA NA

3 14 0 14 0 14 0 NA NA

4 14 0 14 0 14 0 NA NA

0.069 0 16 13 16 16 16 16 NA NA

1 16 1 16 0 16 0 NA NA

2 16 1 16 0 16 0 NA NA

3 16 1 16 0 16 0 NA NA

4 16 0 16 0 16 0 NA NA

0.69 0 6 1 6 6 6 6 NA NA

1 6 2 6 0 6 0 NA NA

2 6 1 6 0 6 0 NA NA

3 6 2 6 0 6 0 NA NA

4 6 0 6 0 6 0 NA NA

6.9 2 0 NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA

2 NA NA NA NA NA NA NA NA

3 NA NA NA NA NA NA NA NA

4 NA NA NA NA NA NA NA NA

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

2 Endpoints at the high treatment level were not statistically analyzed due to high mortality. LOQ=0.014 mg a.i./L

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Page 37 of 89 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitium/multifo

cal inflammation Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Control (<LOQ) 0 14 14 14 14 14 13 14 14

1 14 0 14 0 14 1 14 0

2 14 0 14 0 14 0 14 0

3 14 0 14 0 14 0 14 0

4 14 0 14 0 14 0 14 0

0.069 0 16 16 16 16 16 15 16 16

1 16 0 16 0 16 1 16 0

2 16 0 16 0 16 0 16 0

3 16 0 16 0 16 0 16 0

4 16 0 16 0 16 0 16 0

0.69 0 6 6 6 5 6 4 6 6

1 6 0 6 0 6 2 6 0

2 6 0 6 1 6 0 6 0

3 6 0 6 0 6 0 6 0

4 6 0 6 0 6 0 6 0

6.9 2 0 NA NA NA NA NA NA NA NA

1 NA NA NA NA NA NA NA NA

2 NA NA NA NA NA NA NA NA

3 NA NA NA NA NA NA NA NA

4 NA NA NA NA NA NA NA NA

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

2 Endpoints at the high treatment level were not statistically analyzed due to high mortality. LOQ=0.014 mg a.i./L

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Page 38 of 89 Median male VTG was 9.2 x 10 5 , 4.2 x 10 5 and 8.3 x 10 3 and mean female VTG was 8.3 x 10 5 , 1.2 x

10 6 and 6.4 x 10 5 in the mean-measured <LOQ (control), 0.069 and 6.9 mg a.i./L treatment levels, respectively (Table 19). The male VTG values are unusually high and further discussion of this is presented in the Study Author’s analysis and conclusions section.

Table 19: Plasma Vitellogenin in Fathead Minnow ( Pimephales promelas)

.

Treatment (mg a.i./L)
[mean-measured] Plasma Vitellogenin (VTG)

Males Females

n Median (ng/mL plasma) ±SD 1 n Mean (ng/mL plasma) ±SD 1

Control (<LOQ) 4 9.2 x 10 5 7.0 x 10 5 4 8.1 x 10 5 2.2 x 10 5

0.069 4 4.2 x 10 5 2.7 x 10 5 4 1.2 x 10 6 6.0 x 10 5

0.69 4 8.3 x 10 3 * 9.1 x 10 3 4 6.4 x 10 5 1.0 x 10 6

6.9 2 4 NA NA 4 NA NA

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

1 Standard deviations calculated by the reviewer.

2 Endpoints at the high treatment level were not statistically analyzed due to high mortality. LOQ=0.014 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 39 of 89Mean plasma testosterone and plasma 17ȕ-estradiol in male and females was not measured (Table 20).Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas). Not measured.Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDControl (<LOQ)NA NA NA NA NA NA NA NA NA NA NA NA0.069NA NA NA NA NA NA NA NA NA NA NA NA0.69NA NA NA NA NA NA NA NA NA NA NA NA6.9NA NA NA NA NA NA NA NA NA NA NA NAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.014 mg a.i./L.There were no notable observations with regards to behavior orchanges in appearance, specifically color/banding, ovipositor appearance, or size ofdorsal nape pad in the control or treated groups (Table 21).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 40 of 89Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical Signs at Test TerminationMalesFemalesTypen1Incidence2TypeN1Incidence2Control (<LOQ)Color bars,81None, Papillae present154Territorial aggression4Fatpad, tubercles40.069Color bars,81 None, Papillae present162Territorial aggression4Papillae present1Fatpad, tubercles4None10.69Territorial aggression64 Papillae present62Fatpad, tubercles4None16.9 Fatpad,tubercles11 lethargic 1 1LOQ=0.014 mg a.i./L.1The value reflects the total number of individual fish surviving at test termination2The individual number of fish displaying the reported characteristics/signs was not reported, therefore, the numerical value in the row reflects the number of replicatesreported to be displaying the characteristic.

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Page 41 of 89 B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fertility, fecundity, and VTG. Data were gender specific and analyzed in comparison to the controls.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant effects were detected for p<0.05 with the exception of Shapiro-Wilk’s and Levene’s Tests, whichwere based on p<0.01 (CETIS, Version 1.8.4.20). Survival data were analyzed using a CochranArmitage Trend Step-down test. Male weight, female VTG and GSI were analyzed using ANOVA and a Dunnett’s Multiple comparison test. Fertility and fecundity were analyzed by Dunnett’s T3
Multiple Comparison test. Male VTG and female weight were analyzed by ANOVA and JonckheereTerpstra test. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s test and Levene’s to test for
normality and homogeneity of variances, respectively. If normality and homogeneity tests passed (p>0.01), a parametric analysis was performed using the transformed data. If non-normality or unequal variance were indicated (p<0.01), a non-parametric analysis was performed on the ranks of
the data. These methods appear to be consistent with the methods recommended in the guideline.

Adult survival (both sexes) was 96, 100, 50 and 9% in the <LOQ (control), 0.069, 0.69, and 6.9
mg a.i./L. There was a statistically significant difference in the 0.69 mg a.i./L treatment group compared to the negative control (p<0.05; Cochran-Armitage Trend Step-Down test). Due to the
high mortality at the 6.9 mg a.i./L treatment group, other endpoints were not statistically analyzed (Table 9). There was a statistically significant difference in the 0.69 mg a.i./L treatment level
compared to the negative control for the fecundit y (p<0.05; Dunnett’s Multiple Comparison test; Table 11). There was a statistically significant difference between the 0.69 mg a.i./L treatment level compared to the negative control for the median female body weight (p<0.05; Jonckheere-Terpstra Step-Down test; Table 10). There was a statistically significant difference in median male tubercle
scores at 0.69 mg a.i./L compared to the cont rol (p<0.05; Jonckheere-Terpstra Step-Down Test; Table 12). None of the surviving females were found to have tubercles. There was a statistically
significant difference in the 0.69 mg a.i./L treatment level compared to the negative control for the median male VTG (p<0.05; Jonckheere-Terpstra; Table 19). There were no significant differences
between treated groups and controls for any other endpoint.

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Page 42 of 89 In male fish, there was one incidence of mild testicular degeneration. Otherwise, there were no treatment-related histological effects or differences in gonadal stages in testes in any of the treatment groups (Table 15). In female fish, there was an increase in incidence of minimal to moderate oocyte atresia at the 0.69 mg a.i./L treatment level compared to the control (Table 17). However, the smaller sample size (n=6) prevented further interpretation of this finding. The study
authors did not indicate a treatment-related effect at any test level for gonadal staging. There were
no notable observations with regards to behav ior or changes in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in the control or treated groups (Table 21).

In regards to the male VTG, the study authors did note that the values reported for the control and EPTC treated fish were unusually high. As such, th ey re-analyzed VTG from male fish in replicate A for the control and low and mid treatment groups using an ELISA kit from a different lot than the original analysis. They reported that the results from the second analysis were generally in
agreement with the original values and were not included in the statistical analysis. They also noted
that in the analysis all blanks and standard curves were consistent with historical expectations. The report also noted that there were no obvious explanation for the male VTG levels; there were no changes in the delivery system or dilution water supply or culturing practices and other data from these fish were within historical ranges.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via
Toxstat 3.5. Given that there were only two treatment groups available at test termination, the reviewer concluded that inferences about data exhibiting a trend are limited. Therefore, all endpoints
were analyzed using non-trend statistical tests. All data were tested for normality using ShapiroWilks test and for ho mogeneity of variance usin g Levene’s test. Data which met the assumptions of normality and homogeneity of variance were then analyzed using the parametric Dunnett’s test. Data
which did not meet the parametric assumptions were analyzed using the non-parametric MannWhitney U test.

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EPA MRID Number 48827602

Page 43 of 89 Fertility data was not parametric and were analyzed using Mann-Whitney. All other data wasparametric and were analyzed using Dunnett’s. Male tubercle score was also analyzed using MannWhitney as it is not a continuous endpoint. None of the surviving females were found to have tubercles. The unusually high VTG values reported in the control males makes interpretation of this endpoint difficult and not reliable. Unless otherwise indicated, effects were considered statistically significant at p<0.05. These analyses were conducted using SAS ® (SAS Institute, Cary, NC; version 8.1). Both the study author’s and reviewer relied on the mean-measured concentrations to discuss
effects in this study.

Conclusions: Adult survival of combined sexes was 96, 100, 50 and 9% at <LOQ (control), 0.069, 0.69, and
6.9 mg a.i./L. There was a statistically significant difference in the 0.69 and 6.9 mg a.i./L treatment group compared to the negative control (p<0.05; Fisher’s Exact Test). Due to the high mortality at the 6.9 mg a.i./L treatment group, data were not reported for statistically analysis (Table 9). Male tubercle score was marginally significantly (p=057) reduced by 33% of the control at the 0.69 mg a.i./L treatment level and was approaching significance (p=0.086) with a reduction of 21% at the 0.069 mg a.i./L treatment level. Fecundity (Table 23) was significantly (p<0.05) reduced
75% in the 0.69 mg a.i./L treatment. There were no other significant effects on endpoints. Although
not analyzed statistically, there were no treatment-related histopathologic observations in males. In female fish, there was an increase in incidence of minimal to moderate oocyte atresia at the 0.69 mg a.i./L treatment level compared to the control. However, the smaller sample size (n=6)
prevented further interpretation of this finding. There were no treatment-related effects at any test level for gonadal staging. There were no notable observations with regards to behavior or changes in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape pad in the control or treated groups. There were no notable observations of secondary sex characteristics or clinical signs for any treatment group compared to the controls.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 44 of 89Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with EPTC.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pControl (<LOQ) 40.5 NA 0 NA No0 NA NA NA NA NA0.069 32.0 0.086 -19.67 0.245 No-54.35(NRL)NA NA NA NA NA0.69 26.0 0.057 22.95 0.165 No-99.10(NRL)NA NA NA NA NA6.93NA NA NA NA NA NA NA NA NA NA NAStatistical TestMann-WhitneyDunnett’sNANANANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.NRLDue to the unusually high male VTG values in the control, the results for this endpoint are not reliable for interpretation1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 45 of 89Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with EPTC.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.PMedianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p%Diff.pControl (<LOQ)0 NA 0 NA 0 NA 0 NA No 0 NA NA NA NA NA0.06919.10 0.248 0.00 1.00 0 NA -7.69 0.625 No 47.68 0.625 NA NA NA NA0.69-74.83 <0.001-11.72 1.00 0 NA 23.08 0.080 No -21.07 0.918 NA NA NA NA6.93NA NA NA NA NA NA NA NA NA NA NA NA NA NA NAStatistical TestDunnett’s Mann-Whitney NA Dunnett’s NA Dunnett’s NA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Endpoints at the high treatment level were not statistically analyzed due to high mortality.LOQ=0.014 mg a.i./L.

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Page 46 of 89 Table 24: Growth Endpoints 1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with EPTC.

Treatment (mg a.i./L)
[mean-measured] Body Weight Length

Males Females Males Females

% Diff. p % Diff. p % Diff. p % Diff. p

Control (<LOQ) 0 NA 0 NA 0 NA 0 NA

0.069 -5.56 0.685 2.70 0.896 -2.66 0.688 1.68 0.289

0.69 -5.63 0.679 18.44 0.065 -1.98 0.808 2.45 0.136

6.9 3 NA NA NA NA NA NA NA NA

Statistical Test Dunnett’s Dunnett’s Dunnett’s Dunnett’s

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water)

control.

2 Unless otherwise specified, effects are considered statistically significant at p<0.05.

3 Endpoints at the high treatment level were not statistically analyzed due to high mortality. LOQ=0.014 mg a.i./L.

E. Study Deficiencies

OCSPP 890.1350 validity and performance criteria were met for reported parameters except that dissolved oxygen fell slightly below the recommended 60% of saturation (58%) in one test chamber; the
chambers were cleaned and the levels returned to above the recommended value. In addition, the

unionized ammonia and residual chlorine in the te st water were not reported. The OCSPP 890.1350
performance criteria establish maximum levels for these values and it is unclear if the maximum

recommendations were exceeded.

Additionally, endpoints for fish exposed to the highest treatment level (6.9 mg a.i./L) could not be
evaluated due to mortality; only one male and one female survived at study termination. Therefore, only 2 test treatment levels were analyzed in this study; OCSPP Guideline 890.1350 recommends a

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 47 of 89 minimum of 3 test concentration plus a negative control. There was also significant mortality (50%) at
the 0.69 mg a.i./L treatment level thereby leaving only one test level without the presence of overt toxicity. This limits the ability to interpret the EPTC effects in fish.

While these deviations to the performance criteria were observed, they did not impact the ability to interpret the results of the assay.

F. Reviewer’s Comments

The reviewer’s and the study authors’ results were in agreement regarding effects on survival. Both the study author’s and reviewer’s analysis detected statisti cally significant reductions (p<0.05) in survival atthe 0.69 and 6.9 mg a.i./L treatment level, using either the study author’s analysis method (CochranArmitage Trend test) or the reviewer’s analysis me thod (Fisher’s Exact test). The reviewer’s analysis
additionally detected a marginal significant reduction in median male tubercle score at the 0.069 mg a.i./L treatment level (p=0.053; Dunnett’s test), which the study aut hor did not report. The reviewer’s
conclusions based on the OCSPP 890.1350 flowchart are presented in the Executive Summary and

Conclusions sections of this DER.

In regards to male VTG, given the unusually high values reported in the control, the reviewer determined that the results from this endpoint were not reliable for interpretation. Additionally, in the pathologist’s
report, it was reported that after histological assessment, for one sample submitted as a testis in the control, it was revealed to be a normal ovary. Based on the total number of samples submitted as
female in the control, the reviewer believes this one sample was mis-labeled, as its inclusion as a female would have provided the correct number of female fish gonadal samples in the control.

The method recoveries of EPTC in stock solution fortified at 0.005, 0.500 and 25.0 mg a.i./L was 106 ± 5.7% using a Agilent Zorbax SB-C18 3.5 P column.

The study author reported dissolved oxygen (DO) levels fell to 58% of nominal in the control, therefore
all vessels were cleaned and the DO returned to the required range. Due to the short duration, the deviation is not likely to have a negative impact on the results or interpretation of this study.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

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Page 48 of 89 II. REFERENCES

Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., and Makynen E.A., 2001. Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ

Toxicol Chem 20:1276-1290.

APHA, AWWA, WPCF, 1995. Standard Methods for the Examination of Water and Wastewater. 19th Edition, Washington, DC, 2-23-25.

ASTM, 2005. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM Designation E: 1241-05. American Society for Testing and Materials, 100 Barr Harbor Drive, PO Box C700, West Conshohocken,
Pennsylvania.

Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information SystemTM. User's
Guide. Tidepool Scientific Software, McKinleyville, California.

Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies.

Water Research 1: 21 -29.

Sprague, J.B., 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity.

Water Res. 3: 793-821.

OECD (2009). Fish Short Term Reproduction Assay. OECD Guideline for the testing Chemicals:
Test No. 229. Paris, France. 40 pp

U.S. EPA, 1989. 40CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good Laboratory Practices Standards; Final Rule. Office of the Federal Register, National Archives andRecords Administration. U.S. Government Printing Office, Washington, D.C. U.S. EPA, 2002. Shortterm methods for estimating the chronic toxicity of effluents and receiving waters to marine and

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Page 49 of 89 estuarine organisms. Third edition. Office of Water, U. S. Environmental Protection Agency, Washington, DC 20460. EPA/82 l/R-02/014.

U.S. EPA, 2009. Office of Prevention, Pesticides and Toxic Substances. Endocrine Disrupter Screening Program Test Guideline, OCSPP 890.1350 . Fish Short-Term Reproduction Assay. EPA

40-C-09-007. October 2009. U.S. Environment al Protection Agency. Washington, D.C.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with EPTCEPA MRID Number 48827602Page 50 of 89APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Reviewer’s statistical verification tableEndpoint Monotonic?1Parametric? 890.1350EFEDCommentsFemale bodyweightNA Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectMale bodyweightNA Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectFemale bodylengthNA Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectMale bodylengthNA Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectFemale VTG NAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectMale VTG NANoNANAData not reliableFemale GSI NAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectMale GSI NAYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, noeffectFemaletubercle scoreNA NA NA NA No scoreMale tuberclescoreNA Yes Mann-WhitneyDose 1 p=0.057dunnett’sDose 1 p=0.057EDSP and EFED same conclusions,marginal significant (p=0.057)reductions at the 0.069 mg a.i./Ldoses.Fecundity NA Yes Dunnett’s:Dose 2 p<0.001Dunnett’s:Dose 2 p<0.001EDSP and EFED same conclusions, asignificant (p<0.001) 75% reductionat the 0.69 mg a.i./L dose.Fertility NA No Mann Whitney-Un.s. p>0.05Mann Whitney-Un.s. p>0.05EDSP and EFED same conclusions, noeffectF testosterone NANANANANAM testosterone NANANANANAF estradiol NANANANANAM estradiol NANANANANA

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

-

CONTROL 8 0 8

0.069 8 0 8

---------------------------------------------------------------------------

-

TOTAL 16 0 16

============================================================================
Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 8. Since b is greater than 4.0 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

-

CONTROL 8 0 8

0.69 6 2 8

---------------------------------------------------------------------------

-

TOTAL 14 2 16

============================================================================
Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 6. Since b is greater than 4.0 there is no significant difference between CONTROL and TREATMENT at the 0.05 level.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

-

CONTROL 8 0 8

6.9 2 6 8

---------------------------------------------------------------------------

-

TOTAL 10 6 16

============================================================================
Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 2. Since b is less than or equal to 4.0 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

---------------------------------------------------------------------------

- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 8 0 1 0.069 8 0 2 0.69 8 2 3 6.9 8 6 *

---------------------------------------------------------------------------

-

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION DEAD ALIVE TOTAL ANIMALS

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

-------------------------- ----- ----- --------------

-

CONTROL 1 15 16

0.069 0 16 16

---------------------------------------------------------------------------

-

TOTAL 1 31 32

============================================================================
Critical Fisher's value (16,16,1) (alpha=0.05) is negative. b value is 0. no significant difference

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

-

CONTROL 15 1 16

0.69 6 10 16

---------------------------------------------------------------------------

-

TOTAL 21 11 32

============================================================================
Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 6. Since b is less than or equal to 10.0 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

-------------------------- ----- ----- --------------

-

CONTROL 15 1 16

6.9 1 15 16

---------------------------------------------------------------------------

-

TOTAL 16 16 32

============================================================================
Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 1. Since b is less than or equal to 10.0 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

---------------------------------------------------------------------------

- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 16 1 1 0.069 16 0 2 0.69 16 10 * 3 6.9 16 15 *

---------------------------------------------------------------------------

-

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION DEAD ALIVE TOTAL ANIMALS

-------------------------- ----- ----- --------------

-

CONTROL 1 23 24

0.069 0 24 24

---------------------------------------------------------------------------

-

TOTAL 1 47 48

============================================================================

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Critical Fisher's value (24,24,1) (alpha=0.05) is negative. b value is 0. no significant difference

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

-

CONTROL 23 1 24

0.69 12 12 24

---------------------------------------------------------------------------

-

TOTAL 35 13 48

============================================================================
Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 12. Since b is less than or equal to 18.0 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

-

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

-

CONTROL 23 1 24

6.9 3 21 24

---------------------------------------------------------------------------

-

TOTAL 26 22 48

============================================================================

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 3. Since b is less than or equal to 18.0 there is a significant difference between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

---------------------------------------------------------------------------

- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 24 1 1 0.069 24 0 2 0.69 24 12 * 3 6.9 24 21 *

---------------------------------------------------------------------------

-

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.903 0.203 0.447 0.655 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.39 0.17 0.08 11.96 1.12, 1.65 Dose1 4 1.43 0.11 0.06 7.87 1.25, 1.60 Dose2 3 1.64 0.11 0.06 6.51 1.38, 1.91

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.32 1.28 1.63 . . Dose1 1.43 1.29 1.56 102.70 -2.70 Dose2 1.62 1.55 1.76 118.44 -18.44

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 8 3.50 0.081

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.39 . 1.47 . . . . .

. Dose1 1.43 0.896 1.47 0.870 . . . .

. Dose2 1.64 0.065 1.47 0.882 0.144 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 3.68 0.159

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 1.32 . . Dose1 1.43 0.491 0.808 Dose2 1.62 0.258 0.978

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 8 3.50 0.081

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.39 . -1.39 . . . . .

. Dose1 -1.43 0.896 -1.43 0.417 . . . .

. Dose2 -1.64 0.065 -1.64 0.022 0.144 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 3.68 0.159

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -1.32 . . Dose1 -1.43 0.491 0.192 Dose2 -1.62 0.258 0.022

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose2 Jonckheere Dose2

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.887 0.108 2.910 0.106 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.73 0.18 0.09 4.93 3.44, 4.03 Dose1 4 3.53 0.61 0.31 17.44 2.55, 4.50 Dose2 4 3.52 0.22 0.11 6.34 3.17, 3.88

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 3.69 3.56 3.99 . . Dose1 3.31 3.06 4.43 94.44 5.56 Dose2 3.58 3.22 3.71 94.37 5.63

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 0.38 0.696

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 3.73 . 3.73 . . . . .

. Dose1 3.53 0.685 3.53 0.283 . . . .

. Dose2 3.52 0.679 3.52 0.299 1.000 . . .

.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 2.00 0.368

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 3.69 . . Dose1 3.31 0.346 0.124 Dose2 3.58 0.346 0.190

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 0.38 0.696

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -3.73 . -3.59 . . . . .

. Dose1 -3.53 0.685 -3.59 0.772 . . . .

. Dose2 -3.52 0.679 -3.59 0.804 1.000 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 2.00 0.368

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Level Median MannWhit p-value Jonckheere p-value Ctrl -3.69 . . Dose1 -3.31 0.346 0.876 Dose2 -3.58 0.346 0.810

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.853 0.047 0.965 0.421 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 50.73 1.06 0.53 2.09 49.04, 52.41 Dose1 4 51.58 0.66 0.33 1.28 50.52, 52.63 Dose2 3 51.97 0.51 0.30 0.99 50.69, 53.24

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 50.30 50.00 52.30 . . Dose1 51.35 51.10 52.50 101.68 -1.68 Dose2 52.10 51.40 52.40 102.45 -2.45

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 8 2.23 0.170

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Ctrl 50.73 . 51.37 . . . . .

. Dose1 51.58 0.289 51.37 0.915 . . . .

. Dose2 51.97 0.136 51.37 0.923 0.805 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 3.17 0.205

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 50.30 . . Dose1 51.35 0.230 0.928 Dose2 52.10 0.258 0.967

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 8 2.23 0.170

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -50.73 . -50.73 . . . . .

. Dose1 -51.58 0.289 -51.58 0.103 . . . .

. Dose2 -51.97 0.136 -51.97 0.048 0.805 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Degrees of Freedom TestStat P-value 2 3.17 0.205

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -50.30 . . Dose1 -51.35 0.230 0.072 Dose2 -52.10 0.258 0.033

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose2 Jonckheere Dose2

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.966 0.864 1.433 0.288 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 65.73 1.46 0.73 2.22 63.41, 68.04 Dose1 4 63.98 4.82 2.41 7.54 56.30, 71.65 Dose2 4 64.43 2.80 1.40 4.35 59.97, 68.88

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 65.60 64.10 67.60 . . Dose1 63.20 59.00 70.50 97.34 2.66 Dose2 64.25 61.50 67.70 98.02 1.98

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 0.30 0.749

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 65.73 . 65.73 . . . . .

. Dose1 63.98 0.688 64.20 0.318 . . . .

. Dose2 64.43 0.808 64.20 0.340 0.980 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 1.19 0.551

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 65.60 . . Dose1 63.20 0.346 0.124 Dose2 64.25 0.678 0.232

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 0.30 0.749

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -65.73 . -64.71 . . . . .

. Dose1 -63.98 0.688 -64.71 0.749 . . . .

. Dose2 -64.43 0.808 -64.71 0.782 0.980 . . .

.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 1.19 0.551

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -65.60 . . Dose1 -63.20 0.346 0.876 Dose2 -64.25 0.678 0.768

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.905 0.212 3.622 0.076 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 807500.0 224109.3 112054.7 27.75 450892.0, 1164108 Dose1 4 1192500 599075.7 299537.8 50.24 239236.9, 2145763 Dose2 3 637333.3 1008346 582168.7 158.21 -1867537, 3142203

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 825000.0 580000.0 1000000 . . Dose1 1100000 570000.0 2000000 147.68 -47.68 Dose2 110000.0 2000.00 1800000 78.93 21.07

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

2 8 0.72 0.518

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 807500.0 . 1000000 . . . . .

. Dose1 1192500 0.625 1000000 0.746 . . . .

. Dose2637333.3 0.918 637333.3 0.465 0.519 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 1.63 0.443

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 825000.0 . . Dose1 1100000 0.484 0.813 Dose2 110000.0 0.612 0.368

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 8 0.72 0.518

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Ctrl -807500 . -807500 . . . . .

. Dose1-1192500 0.625 -954571 0.446 . . . .

. Dose2 -637333 0.918 -954571 0.486 0.519 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 1.63 0.443

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -825000 . . Dose1 -1100000 0.484 0.187 Dose2 -110000 0.612 0.632

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.947 0.597 17.716 <.001 USE NON-PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 920000.0 697471.6 348735.8 75.81 -189833, 2029833 Dose1 4 420000.0 274469.2 137234.6 65.35 -16741.7,856741.7 Dose2 4 8252.50 9099.29 4549.65 110.26 -6226.51,22731.51

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 885000.0 210000.0 1700000 . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Dose1 300000.0 250000.0 830000.0 45.65 54.35 Dose2 7445.00 120.00 18000.00 0.90 99.10

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 4.45 0.045

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 920000.0 . 920000.0 . . . . .

. Dose1420000.0 0.229 420000.0 0.081 . . . .

. Dose2 8252.50 0.028 8252.50 0.009 0.407 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 7.73 0.021

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 885000.0 . . Dose1 300000.0 0.494 0.193 Dose2 7445.00 0.067 0.003

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 4.45 0.045

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -920000 . -449418 . . . . .

. Dose1 -420000 0.229 -449418 0.960 . . . .

. Dose2-8252.50 0.028 -449418 0.971 0.407 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 7.73 0.021

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -885000 . . Dose1 -300000 0.494 0.807 Dose2 -7445.00 0.067 0.997

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.904 0.207 1.345 0.314 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 13.00 0.82 0.41 6.28 11.70, 14.30 Dose1 4 12.00 2.16 1.08 18.00 8.56, 15.44 Dose2 3 16.00 1.73 1.00 10.83 11.70, 20.30

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 13.00 12.00 14.00 . . Dose1 11.50 10.00 15.00 92.31 7.69 Dose2 15.00 15.00 18.00 123.08 -23.08

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 8 5.22 0.035

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 13.00 . 13.45 . . . . .

. Dose1 12.00 0.625 13.45 0.733 . . . .

. Dose2 16.00 0.080 13.45 0.757 0.032 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 5.60 0.061

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 13.00 . . Dose1 11.50 0.410 0.153 Dose2 15.00 0.095 0.898

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Numerator df Denominator df F-stat P-value 2 8 5.22 0.035

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -13.00 . -12.50 . . . . .

. Dose1 -12.00 0.625 -12.50 0.746 . . . .

. Dose2 -16.00 0.080 -16.00 0.028 0.032 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 5.60 0.061

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -13.00 . . Dose1 -11.50 0.410 0.847 Dose2 -15.00 0.095 0.102

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose2 Jonckheere >highest dose (no sign. differences)

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.909 0.210 2.800 0.113 USE PARAMETRIC TESTS

*****************************************************************************

***

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.53 0.32 0.16 20.99 1.02, 2.03 Dose1 4 1.23 0.29 0.14 23.45 0.77, 1.68 Dose2 4 1.88 0.17 0.09 9.11 1.60, 2.15

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.55 1.20 1.80 . . Dose1 1.15 1.00 1.60 80.33 19.67 Dose2 1.85 1.70 2.10 122.95 -22.95

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 5.93 0.023

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.53 . 1.54 . . . . .

. Dose1 1.23 0.245 1.54 0.620 . . . .

. Dose2 1.88 0.165 1.54 0.655 0.018 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 6.39 0.041

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 1.55 . . Dose1 1.15 0.278 0.093 Dose2 1.85 0.225 0.896

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 5.93 0.023

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.53 . -1.38 . . . . .

. Dose1 -1.23 0.245 -1.38 0.852 . . . .

. Dose2 -1.88 0.165 -1.88 0.060 0.018 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 6.39 0.041

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -1.55 . . Dose1 -1.15 0.278 0.907 Dose2 -1.85 0.225 0.104

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.00 0.00 0.00 . . , . Dose1 4 0.00 0.00 0.00 . . , . Dose2 4 0.00 0.00 0.00 . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.00 0.00 0.00 . . Dose1 0.00 0.00 0.00 . . Dose2 0.00 0.00 0.00 . .

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value . . . 9 . . . . . .

.

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . .

. Dose1 0.00 . . . . . . .

. Dose2 0.00 . . . . . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.00 . . Dose1 0.00 1.000 .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Dose2 0.00 1.000 .

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose1

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value . . . 9 . . . . . .

.

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . .

. Dose1 0.00 . . . . . . .

. Dose2 0.00 . . . . . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.00 . . Dose1 0.00 1.000 . Dose2 0.00 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1 Jonckheere Dose1

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) )

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.965 0.850 3.440 0.078 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 39.75 5.12 2.56 12.89 31.60, 47.90 Dose1 4 31.50 1.91 0.96 6.08 28.45, 34.55 Dose2 4 26.75 5.62 2.81 21.01 17.81, 35.69

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 40.50 33.00 45.00 . . Dose1 32.00 29.00 33.00 79.25 20.75 Dose2 26.00 22.00 33.00 67.30 32.70

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 8.44 0.009

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 39.75 . 39.75 . . . . .

. Dose1 31.50 0.053 31.50 0.017 . . . .

. Dose2 26.75 0.005 26.75 0.002 0.343 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 7.03 0.030

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Level Median MannWhit p-value Jonckheere p-value Ctrl 40.50 . . Dose1 32.00 0.096 0.019 Dose2 26.00 0.080 0.003

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose1

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 8.44 0.009

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -39.75 . -32.67 . . . . .

. Dose1 -31.50 0.053 -32.67 0.989 . . . .

. Dose2 -26.75 0.005 -32.67 0.993 0.343 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 7.03 0.030

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -40.50 . . Dose1 -32.00 0.096 0.981 Dose2 -26.00 0.080 0.997

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.971 0.916 0.482 0.632 USE PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 22.25 2.99 1.49 13.42 17.50, 27.00 Dose1 4 26.50 5.00 2.50 18.87 18.54, 34.46 Dose2 4 5.60 3.09 1.55 55.23 0.68, 10.52

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 23.00 18.00 25.00 . . Dose1 26.00 21.00 33.00 119.10 -19.10 Dose2 5.50 2.00 9.40 25.17 74.83

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 33.67 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 22.25 . 24.38 . . . . .

. Dose1 26.50 0.248 24.38 0.851 . . . .

. Dose2 5.60 <.001 5.60 <.001 <.001 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

2 8.22 0.016

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 23.00 . . Dose1 26.00 0.281 0.906 Dose2 5.50 0.066 0.046

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 33.67 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -22.25 . -18.12 . . . . .

. Dose1 -26.50 0.248 -18.12 0.959 . . . .

. Dose2 -5.60 <.001 -18.12 0.971 <.001 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 8.22 0.016

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -23.00 . . Dose1 -26.00 0.281 0.094 Dose2 -5.50 0.066 0.954

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - EPTC ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.732 0.002 6.115 0.021 USE NON-PARAMETRIC TESTS

*****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 96.00 1.41 0.71 1.47 93.75, 98.25 Dose1 4 96.00 1.83 0.91 1.90 93.09, 98.91 Dose2 4 84.75 20.39 10.19 24.05 52.31, 117.19

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 96.50 94.00 97.00 . . Dose1 96.00 94.00 98.00 100.00 0.00 Dose2 93.00 55.00 98.00 88.28 11.72

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 1.20 0.344

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 96.00 . 96.00 . . . . .

. Dose1 96.00 1.000 96.00 0.583 . . . .

. Dose2 84.75 0.344 84.75 0.134 0.408 . . .

.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 0.07 0.966

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 96.50 . . Dose1 96.00 1.000 0.559 Dose2 93.00 1.000 0.470

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

*****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 1.20 0.344

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -96.00 . -92.25 . . . . .

. Dose1 -96.00 1.000 -92.25 0.754 . . . .

. Dose2 -84.75 0.344 -92.25 0.787 0.408 . . .

.

*****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 0.07 0.966

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602

Level Median MannWhit p-value Jonckheere p-value Ctrl -96.50 . . Dose1 -96.00 1.000 0.441 Dose2 -93.00 1.000 0.530

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

Male Tubercle Score (Mann-Whitney)



### Obs conc endpoint 1 

### 0 39 2 

### 0 33 3 

### 0 42 4 

### 0 45 5 

### 1 33 6 

### 1 33 7 

### 1 31 8 

### 1 29 9 

### 2 30 10 

### 2 33 11 

### 2 22 12 

### 2 22 13 

### 3

. 14 

### 3

. 15 

### 3

. 16 

### 3

.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



### The NPAR1WAY Procedure

Wilcoxon Scores (Rank Sums) for Variable endpoint Classified by Variable conc

conc N Sum of Scores Expected Under H0 Std Dev Under H0 Mean Score 0 

### 4 40.50 26.0 5.773503 10.1250 1 

### 4 23.00 26.0 5.773503 5.7500 2 

### 4 14.50 26.0 5.773503 3.6250 Average scores were used for ties.

Kruskal-Wallis Test Chi-Square 

### 7.0300 DF 

### 2Asymptotic Pr > ChiSquare 

### 0.0297

Exact Pr >= ChiSquare 

### 0.0152

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



### The NPAR1WAY Procedure

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



### The NPAR1WAY Procedure

Wilcoxon Scores (Rank Sums) for Variable endpoint Classified by Variable conc

conc N Sum of Scores Expected Under H0 Std Dev Under H0 Mean Score 0 

### 4 25.0 18.0 3.380617 6.250 1 

### 4 11.0 18.0 3.380617 2.750 Average scores were used for ties.

Wilcoxon Two-Sample Test Statistic (S) 

### 25.0000

Normal Approximation Z 

### 1.9227 One-Sided Pr > Z 

### 0.0273 Two-Sided Pr > |Z| 

### 0.0545

t Approximation One-Sided Pr > Z 

### 0.0480 Two-Sided Pr > |Z| 

### 0.0959

Exact Test One-Sided Pr >= S 

### 0.0429 Two-Sided Pr >= |S - Mean| 

### 0.0857
Z includes a continuity correction of 0.5.

Kruskal-Wallis Test Chi-Square 

### 4.2875 DF 

### 1Pr > ChiSquare 

### 0.0384

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



### The NPAR1WAY Procedure

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



### The NPAR1WAY Procedure

Wilcoxon Scores (Rank Sums) for Variable endpoint Classified by Variable conc

conc N Sum of Scores Expected Under H0 Std Dev Under H0 Mean Score 0 

### 4 25.50 18.0 3.422614 6.3750 2 

### 4 10.50 18.0 3.422614 2.6250 Average scores were used for ties.

Wilcoxon Two-Sample Test Statistic (S) 

### 25.5000

Normal Approximation Z 

### 2.0452 One-Sided Pr > Z 

### 0.0204 Two-Sided Pr > |Z| 

### 0.0408

t Approximation One-Sided Pr > Z 

### 0.0400 Two-Sided Pr > |Z| 

### 0.0801

Exact Test One-Sided Pr >= S 

### 0.0286 Two-Sided Pr >= |S - Mean| 

### 0.0571
Z includes a continuity correction of 0.5.

Kruskal-Wallis Test Chi-Square 

### 4.8018 DF 

### 1Pr > ChiSquare 

### 0.0284

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



### The NPAR1WAY Procedure

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with EPTC

EPA MRID Number 48827602



### The NPAR1WAY Procedure

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-13-2012 (MRID 48616406)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Rebecca L. Bryan, B.S. Date: 1/19/2012

Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 1/23/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 1/27/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 1/27/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).In vivo Hershberger Assay (2011)/ Page 1of13 EPTC/041401 

### Primary Reviewer: Sheila Healy, Ph.D. 

### Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D. 

### Health Effects Division

DAT A EVALUATION RECORD OCSPP 890.1400/ OECD 441



### STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441



### PC CODE: 041401 

### DP BARCODE: D397 

### l 77



### TXR#: 0051762 

### CAS#: 759-94-4



### TEST MATERIAL (PURITY): EPTC ( > 99.9% a.i.)



### SYNONYMS: S-ethyl N,N -dipropylcarbamothioate



### CITATION: Zorrilla, L. 

### (2011). The Hershberger Assay with EPTC in Castrated CRL:CD(SD) IGS Rats. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C201-200, November 4, 2011. MRID 48616406. Unpublished.



### SPONSOR: Gowan Company, P.O. Box 5569
, Yuma
, AZ



### TEST ORDER#: EDSP-041401-64



### EXECUTIVE SUMMARY: 

### In a Hershberger assay (MRID 48616406) screening for androgenic activity, EPTC ( > 99.9% a.i., lot # Pl 105096) in corn oil was administered daily via oral gavage to groups of 60- to 61-day old
, castrated male Sprague Dawley rats (8 / group) at dose levels of 0 (vehicle), 50
, or 250 mg / kg / day. An androgenic positive control group consisting of 8 castrated rats exposed to 0.4 mg / kg / day testosterone propionate (TP) by subcutaneous (s.c.) injection was also included as a positive control.

To screen for potential anti-androgenic activity, EPTC ( > 99

. 9% a.i., lot # P 1105096) in corn oil

was administered daily via ora l gavage to 60- to 61-day old, castrated male Sprague Dawley rats (8 / group) at dose levels of 50
, 150
, or 250 mg / kg / day in conjunction with a daily dose of reference androgen TP at 0.4 mg / kg / day by s.c. injection. The control group consisted of eight castrated male rats dosed daily with TP (0.4 mg / kg / day) by s.c. injection, and the anti-androgenic positive control group consisted of eight castrated male rats dosed orally with flutamide (FT) in

corn oil at 3 mg / kg / day in conjugation with a daily s.c. dose of TP (0.4 mg / kg / day)

.

For both components of the assay, the animals were dosed for I 0 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues.

One positive control (TP only) rat died after the first day of dosing due to a gavage error. All other animals survived until scheduled termination

. There were no treatment-related clinical signs of toxicity.In vivo Hershberger Assay (2011)/ Page 2 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441



### In the androgen agonist a ssay, body weights for the 50 and 250 mg/kg/day EPTC-treatment groups were comparable to controls throughout the duration of the assay. At 250 mg/kg/day, overall (Days 1-11) body wei ght gains were decreased (p ≤ 0.05) by 40%. Animals dosed with TP had a 10% increase (p ≤ 0.05) in body weights on Day 11 a nd a 69% increase in overall body weight gains compared to controls. Animal s in the positive control (TP) group had increases (p ≤ 0.05) in accessory sex organ weights as follo ws: 1578% in seminal vesicles; 1542% in ventral prostate; 259% in the levator ani-bulbocavernosus (LABC) muscle complex; 550% in Cowper’s gland; and 70% in glans penis. Th e maximum guideline-specified %CV was slightly exceeded for the seminal vesicle in the 250 mg /kg/day (43% vs. 40%) group, but all other %CV values were less than the maximum permissible values.

In the anti-androgen assay, body weights in the 50 and 150 mg/kg/day groups were comparable to controls throughout the duration of the assa y. At 250 mg/kg/day, overall body weight gains were decreased (p ≤ 0.05) by 34%. For the positive control group (TP + FT), overall body weight gains were decreased (not significant, NS) by 20% . At 50 mg/kg/day of EPTC, the weights of seminal vesicles were decreased by 23% (p ≤ 0.05). The LABC weights were decreased by 10% (p ≤ 0.05) at 150 mg/kg/day and by 16% (p ≤ 0.05) at 250 mg/kg/day. Animals dosed with TP + FT (positive control) had decreases (p ≤ 0.05) in accessory sex organ weights as follows: 88% in seminal vesicles; 93% in ventra l prostate; 55% in LABC; and 65% in Cowper’s gland. The maximum guideline-specified %CV was slightly exceeded for the glans penis in the 250 mg/kg/day (19% vs. 17%) group. All other %CV values were less than the maximum permissible values.

Based on the decreases in overall body weight gain observed at the high-dose (250 mg/kg/day) in both the androgenic and anti-androgenic assays, the dosing was considered to be adequate.

Statistically significant changes in organ weights were not seen in two or more of the five androgen sensitive tissues. EPTC was negative for androgenicity and anti-androgenicity in the Hershberger assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Hershberger assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.In vivo Hershberger Assay (2011)/ Page 3 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratory Systems, Inc. Location: Durham, NC Study Director: L. Zorrilla Other Personnel: Not provided Study Period: July 15-26, 2011



### 2. Test Substance: EPTC Description: Off white solid Source: Gowan Company (commerc ial technical product) Lot/Batch #: P1105096, expires February 2, 2015 Purity: >99.9 % a.i. Stability: Stable in the vehicle for up to 25 days at 1-10°C (Smithers Viscient Inc., Study No. 12791-6162) CAS #: 759-94-4 Structure:



### 3. Reference Androgen: Testosterone propionate (TP) Supplier: Sigma-Aldrich Company (St. Louis, MO) Batch #: 048K1328 Purity: 100.2 % CAS # : 57-85-2


### 4. Reference Anti-androgen: Flutamide (FT) Supplier: Sigma-Aldrich (St. Louis, MO) Lot #: 107K1293 Purity: >99 % CAS # : 13311-84-7



### 5. Solvent/Vehicle Control: Corn Oil Supplier: MP Biomedicals, LLC (Solon, OH) Lot/Batch #: 7862K Rationale (if other than water) : Not provided Final concentration: Not applicable



### 6. Test Animals: Species: Rat (castrated males) Strain: Sprague Dawley (Crl:CD ® [SD] IGS) Age/weight at dose initiation: Post-natal day (PND) 60/61; 249.2-303.7 g Source: Charles River Laboratories International, Inc., Raleigh, NC Housing: Rats were housed up to 2/cage in polyc arbonate cages with absorbent heattreated hardwood bedding (Northeastern Products Corp., Warrensburg, NY). Diet: Teklad Global 16% Protein Rodent Di et (Teklad Diets, Madison, WI), ad libitum ; low phytoestrogen with total geni stein equivalents = 8.2 μg/g diet Water: Reverse osmosis tap water , ad libitum Environmental conditions: Temperature: 22-25oC Humidity: 34-60% Air changes: Not reported Photoperiod: 12 hrs light/12 hrs dark Acclimation period: At least 8 days S N CH 3

CH 3 O

CH

3In vivo Hershberger Assay (2011)/ Page 4 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441



### B. STUDY DESIGN

1. In-life dates: Start: 7/15/2011 End: 7/26/2011

2. Study Design: The current Hershberger Assay was c onducted to screen for both potential androgenic and anti-androgenic activity. In the test for androgeni c activity, EPTC was administered daily via oral gavage to groups of castrated male rats. Positive androgenic activity is defined as a significant increase in tw o or more target organ weights compared to the vehicle control group. The positive contro l group for androgenic ac tivity is a group of castrated male rats dosed by s.c. injection with TP at 0.4 mg/kg/day. In the test for antiandrogenic activity, EPTC was admi nistered daily via oral gava ge to groups of castrated male rats in conjunction with TP at 0.4 mg/kg/day by s.c. injection. Anti-androgenic activity is indicated by a statistically signifi cant decrease in two or more target organ weights of the treated groups (test substance + TP) compared to the TP-only control group. The positive control group for anti-androgenic activity is a gr oup of castrated male rats dosed by s.c. injection with TP (0.4 mg/kg/day) in conjunction with an oral dose of FT (3.0 mg/kg/day). For both assays, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the fi nal dose administration for measurement of target organ weights.

3. Study Schedule: Following an 8-day acclimation period at the test facility, castrated male rats (surgical manipulation performed by Supplie r) were administered the test substances from PND 60/61 to PND 69/70. Rats were euthanized on PND 70/71, approximately 24 hours after the last dose and necropsied fo r target organ weight measurements.

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical anal ysis indicated that th ere were no significant differences in group means at study initiation.

TABLE 1. Study design a Test group Dose (mg/kg/day) # of Males Androgen Agonist Assay Vehicle control (negative control) 0 8 Low EPTC 50 8 High EPTC 250 8 Testosterone propionate (TP) (s.c.), positive control b 0.4 8

Anti-Androgen Assay Testosterone propionate (negative control, s.c.) b 0 + 0.4 8 Low EPTC +TP 50 + 0.4 8 Mid EPTC +TP 150 + 0.4 8 High EPTC +TP) 250 + .04 8 Flutamide (oral gavage) + TP (s.c.), positive control 3 = 0.4 8

a Data were obtained from page 15 of the study report. b Same animals dosed for both androgen agonist (served as positive control) and anti-androgen (served as vehicle control) assays. This did not affect the outcome or conclusions of this study.In vivo Hershberger Assay (2011)/ Page 5 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441


### 5. Dose Selection Rationale: The EPTC dose levels were se lected based on th e results of a subchronic neurotoxicity study and a developmental toxicity range-finding study (study report numbers not provided). In the ne urotoxicity study, Alpk:APfSd rats were administered EPTC in the diet at dose levels of 100, 500, or 2500 ppm (equivalent to 7.9, 34.9, and 195.1 mg/kg/day) for 13 weeks. It wa s stated that bodyweights for the 2500 ppm animals were slightly reduced ( ↓ 4% in males and ↓ 3.3% in females) during Week 2. In the developmental toxicity range-finding study, female rats were administered EPTC in corn oil by daily oral gavage at dose levels of 50, 90, 160, 280, or 500 mg/kg/day during gestation days 6-15. It was stated that all 500 mg/kg/day animals died during dosing. At 160 mg/kg/day and above, body weight gains were decreased by 29-62%, food consumption was reduced, and clinical signs (yellow urogen ital matting and staining and red matter around the nasal area) were observed. Based on these results, doses of ≤ 250 mg/kg/day were used for both the androgen agonist and anti-androgen assays.

6. (a) Dose Preparation: Dose formulations were prep ared once prior to initiation of treatment by suspending the appropriate am ounts of test substance in corn oil

. Flutamide and TP were prepared once pr ior to initiation of treatment by mixing in corn oil. Homogeneity and concentration analyses were performed on all dose formulations. No stability analyses were perf ormed with the current study.

In a previous storage stability study 1 

### , EPTC (lot number not repor ted) was determined to be stable in corn oil for up to 25 days at 1-10°C.

( b) Dose Analysis

Results

Homogeneity (%CV): 1.14-5.01%

Stability (% of nominal): Data not provided

Concentration (% of nominal): 101-104%
The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

7. Dosage administration: Test formulations of EPTC were administered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and FT was administered via oral gavage at 5 mL/kg bw. Dose volumes were adjusted daily based on the concurrent body weight measurement.

8. Statistics: Final body weights, body weight gains and accessory sex organ weights were analyzed using studentized residual plots for outliers and Levene’s te st for homogeneity of variance. Heterogeneous data (Cowper’s gl and, anti-androgen assay) were transformed (logarithm, square root, multiplicative inverse transformations) and re-analyzed using

1 Reibach, R. 2011b. Storage Stability of EPTC in Corn Oil. Unpublished study re port prepared by Smithers Viscient Inc., Study No. 12791-6162.In vivo Hershberger Assay (2011)/ Page 6 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441 

### Kruskal-Wallis and Dunn’s test. Final body wei ghts, body weight gains, and accessory sex organ weights were analyzed using one- way ANOVA followed by Dunnett’s t-tests. The positive controls were compared to the vehicle controls using the t-test. Significance was denoted at p ≤ 0.05. The statistical analyses were consid ered adequate. Data were analyzed using SAS version 9.2 software.

C. METHODS

1. Clinical Examinations: Animals were checked for mort ality or moribundity twice daily on weekdays and once daily on weekends. Rats were also checked for clinic al signs of toxicity prior to dose administration and at termination.

2. Body Weight: Animals were weighed at randomiza tion, daily prior to dosing throughout the dosing period, and at termination.

3. Food Consumption (Optional): Food consumption was not determined.

4. Serum Hormone Measurements (Optional): Serum hormone measurements were not conducted.

5. Dissection and Measurement of Tissue and Organ Weights: On PND 70-71 (approximately 24 hours after the final administra tion of the test substa nce), all rats were weighed, anesthetized by CO 2 

### inhalation followed by cervical dislocation and subjected to a gross necropsy. The five mandatory androgen- dependent organs (ventr al prostate, seminal vesicles with coagulating gla nds and fluid, LABC, Cowper’s gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400).

II. RESULTS

A. OBSERVATIONS

1. Mortality: One positive control (TP only) rat died after the first day of dosing due to a gavage error. All other animals survived until scheduled termination.

2. Clinical signs of toxicity: No treatment-related clinical si gns of toxicity were observed in animals for any dose groups.

In the androgen agonist assay, one 250 mg/kg/ day rat was observed w ith rough coat and red discoloration of nose fur. In the anti -androgen assay, observations included one 50 mg/kg/day rat with a facial scab (Days 7-8) a nd also red discoloration of nose fur (Day 11) and one 250 mg/kg/day rat with re d discoloration of nose fur (Day 11). Salivating rats were observed after dosing on Day 3/4 for the 250 mg/kg/day group (3 rats) of the androgen agonist assay, and the 50 mg/kg/ day (1 rat) and 150 mg/kg/day (2 rats) groups of the antiandrogen assay. These observations were not considered treatment-related.In vivo Hershberger Assay (2011)/ Page 7 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441


### B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gains for the androgen agonist assay are presented in Table 2. Body weights for the 50 and 250 mg/kg/day treatment groups were comparable to controls throughout the duration of the assay. At 250 mg/kg/day, overall (Days 1- 11) body weight gains were decreased (p ≤ 0.05) by 40%. Animals dosed with TP had increased (p ≤ 0.05) body weights on Day 11 ( ↑ 10%) and increased overall body weight gains ( ↑ 69%) compared to controls.

TABLE 2. Selected Group Mean (±SD) Body Weight s and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.4) b EPTC (50) EPTC (250)

N Mean SD N Mean SD N Mean SD N Mean SD 1 8 275.1 15.1 8 277.0 18.9 8 279.8 17.5 8 278.4 16.2 4 8 282.7 20.3 8 297.2 20.2 8 291.1 23.8 8 284.2 15.7 7 8 296.3 18.6 8 317.9 25.9 8 306.4 26.5 8 297.4 19.0 11 8 315.7 20.6 8 345.9* ( ↑ 10) 33.9 8 323.2 31.7 8 303.0 31.4

B WG (Days 1-11) 8 40.7 20.6 8 68.9* ( ↑ 69) 19.5 8 43.5 15.0 8 24.6* ( ↓ 40) 23.6

a Data were obtained from Table 4 on page 20 and Appendix V on page 74 of the study report. Percent differences from controls (calculated by the reviewers) are included in parentheses. b The positive control group for the androgen agonist is same as the vehicle control for the anti-androgen. This did not affec t the outcome or conclusions of this study. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05.



### Body weight and body weight gain data for the anti-androgen a ssay are presented in Table 3. Body weights in the 50 and 150 mg/kg/da y groups were comparable to controls throughout the duration of the assay. At 250 mg/kg/day, overall body weight gains were decreased (p ≤ 0.05) by 34%. For the positive control group (TP + FT), overall body weight gains were decreased (NS) by 20%.In vivo Hershberger Assay (2011)/ Page 8 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441

TABLE 3. Selected Group Mean (±SD) Body Weights a nd Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control

+ TP (0.4) b Positive Control TP + FT (0.4 + 3.0) EPTC+TP 50 + 0.4) EPTC+TP (150 + 0.4) EPTC+TP (250 + 0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 8 277.0 18.9 8 277.6 17.7 8 278. 2 16.8 8 277.7 16.2 8 278.9 12.1 4 8 297.2 20.2 8 293.2 20.8 8 296. 2 18.6 8 289.5 18.7 8 291.7 13.6 7 8 317.9 25.9 8 309.0 23.1 8 318. 0 22.7 8 313.5 23.4 8 311.9 16.0 11 8 345.9 33.9 8 332.8 25.7 8 345. 9 28.4 8 339.5 22.5 8 324.1 16.7 B WG (days 1-11) 8 68.9 19.5 8 55.2 ( ↓ 20) 9.3 8 67.6 15.6 8 61.8 15.5 8 45.3* ( ↓ 34) 12.3

a Data were obtained from Table 5 on page 21 and Appendix V on page 74-75 of the study report. Percent differences from controls (calculated by the reviewers) are included in parentheses. b The vehicle control for the anti-androgen is same as the positiv e control group for the androgen agonist. This did not affec t the outcome or conclusions of this study. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05



### C. FOOD CONSUMPTION (Optional): Food consumption was not determined.

D. SERUM HORMONE CONCE NTRATIONS (Optional): Serum hormone concentrations were not determined.

E. ORGAN WEIGHTS: Accessory sex organ weights fo r the androgen agonist assay are presented in Table 4. There were also no trea tment-related effects in any of the accessory sex organs of animals dosed with EPTC. Animals in the TP group had increases (p ≤ 0.05) in accessory sex organ weights as follows: 1578% in seminal vesicles; 1542% in ventral prostate; 259% in LABC; 550% in Cowpe r’s gland; and 70% in glans penis.

The maximum guideline-specified %CV was sligh tly exceeded for the seminal vesicle %CV in the 250 mg/kg/day EPTC group (43% vs. 40%). All other %CV values were less than the maximum permissible values.In vivoHershberger Assay (2011)/ Page 9 of 13EPTC/041401OCSPP 890.1400/ OECD 441TABLE 4. Accessory Sex Organ Weights (g) fromAndrogen Agonist Assay in Sprague-Dawley RatsaOrganDose (mg/kg/day)Vehicle controlEPTC (50)EPTC (250)Positive ControlVehicle + TP (0.4)bN Mean SD CV(%)N Mean SD CV(%)N Mean SD CV(%)N Mean SD CV(%)Seminalvesicles8 0.0427 0.0101 24 8 0.0357 0.007521 8 0.0372 0.0158 43 8 0.7163*(↑1578)0.1581 22Ventralprostate8 0.0115 0.0050 43c8 0.0089 0.0029 33 8 0.0080 0.0028 35 8 0.1888*(↑1542)0.0473 25LABC8 0.1513 0.0242 16 8 0.1689 0.026416 8 0.1411 0.0265 19 8 0.5431*(↑259)0.0529 10Cowper’sglands8 0.0064 0.0020 31 8 0.0062 0.001220 8 0.0059 0.0020 34 8 0.0416*(↑550)0.0098 24Glans penis8 0.0503 0.0086 17 8 0.0506 0.007415 8 0.0507 0.0096 19 8 0.0855*(↑70)0.0075 9a Data were obtained from Table 6 on page 23 and Appendix VI on page 77 of the study report. Percent differences from controlswere calculated by the reviewers andincluded in parentheses.b The positive control group for the androgenagonist is same as the vehicle controlfor the anti-androgen. This did not affect the outcome or conclusions of this study.c The %CV exceeds the maximum acceptable %CV.N Number of animals in the groupSD Standard DeviationCV Coefficient of VariationNA Not applicable* Significantly different from controls at p≤0.05In vivo Hershberger Assay (2011)/ Page 10 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441



### Accessory sex organ weights for the anti-andr ogen assay are presente d in Table 5. At 50 mg/kg/day, the weights of seminal ve sicles were decreased by 23% (p ≤ 0.05). The LABC weights were decreased by 10% (p ≤ 0.05) at 150 mg/kg/day and 16% (p ≤ 0.05) at 250 mg/kg/day. Animals dosed with TP + FT (positive control) had decreases (p ≤ 0.05) in accessory sex organ weights as follows: 88% in seminal vesicles; 93% in ventral prostate; 55% in LABC; and 65% in Cowper’s gland.

The maximum guideline-specified CV was slight ly exceeded for the glans penis in the 250 mg/kg/day (19% vs. 17%) group. All other CV values were less than the maximum permissible values.In vivoHershberger Assay (2011)/ Page 11 of 13EPTC/041401OCSPP 890.1400/ OECD 441TABLE 5. Accessory Sex Organ Weights (g) from Anti-Androgen Agonist Assayin Sprague-Dawley RatsaOrganDose (mg/kg/day)Vehicle Control+ TP (0.4)bEPTC+TP(50+0.4)EPTC+TP(150+0.4)EPTC+TP(250+0.4)TP + FT (0.4+3.0)Positive ControlN Mean SD CV(%)N Mean SD CV(%)N Mean SD CV(%)N Mean SD CV(%)N Mean SD CV(%)Seminalvesicles8 0.7163 0.1581 22 8 0.5486*(↓23)0.1383 25 8 0.5766 0.0674 12 8 0.6224 0.1470 24 8 0.0895*(↓88)0.0331 37Ventralprostate8 0.1888 0.0473 25 8 0.1715 0.0319 19 8 0.1612 0.0247 15 8 0.1653 0.0260 16 8 0.0137*(↓93)0.0051 37LABC8 0.5431 0.0529 10 8 0.4956 0.0501 10 8 0.4912*(↓10)0.0484 10 8 0.4537*(↓16)0.0340 7 8 0.2464*(↓55)0.0108 4Cowper’s glands8 0.0416 0.0098 24 8 0.0417 0.0059 14 8 0.0396 0.0039 10 8 0.0387 0.0066 17 8 0.0146*(↓65)0.0033 23Glans penis 8 0.0855 0.0075 9 8 0.0948 0.0130 14 8 0.0990 0.0096 10 8 0.0911 0.0169 19c8 0.0787 0.0086 11a Data were obtained from Table 7 on page 24 and Appendix VI on page 78 of the study report. Percent differences from controlswere calculated by the reviewers andincluded in parentheses.b The vehicle control for the anti-androgen is same as the positive control group for the androgen agonist. This did not affect the outcome or conclusions of this study.c The %CV exceeds the maximum acceptable %CV.N Number of animals in the groupSD Standard DeviationCV Coefficient of VariationNA Not applicable* Significantly different from controls at p≤0.05In vivo Hershberger Assay (2011)/ Page 12 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: In the agonist assay, the final mean body weight of animals administered 250 mg/ kg/day EPTC was 96% of the vehi cle control as a result of the mean body weight gain during the dosing inte rval being statistica lly significantly lower than the control. EPTC administered at dose levels of 50 and 250 mg/kg/day did not increase androgen dependent tissue weights.

In the androgen-antagonist assay, final m ean body weights of animals administered 250 mg/kg/day EPTC plus TP were 94% of the control (vehicle and TP ) as a result of the mean body weight gain during the dosing interval being statistically significantly decreased compared to control. A significant reduction in seminal vesicle weight was observed in animals administered 50 mg/kg/day EPTC plus TP when compared to the TP-control group, however, no change in the weight of this ti ssue occurred in the two higher dose levels (150 and 250 mg/kg/day) to sugge st a biological eff ect of the change in this tissue with administration of EPTC plus TP. EPTC co-a dministered with TP caused a statistically significant dose-dependent decrease in LA BC weights (10% and 17% for the 150 and 250 mg/kg/day dose groups, respectively) as compared to the control group, however no other androgen-dependent weights were redu ced at these dose levels, therefore, the Hershberger Bioassay is negative for EPTC at doses up to 250 mg/kg/day.

Oral administration of EPTC of up to 250 mg/ kg/day for 10 consecutive days, to castrated adult rats, did not result in androgen agonist or antagonist activity and 5- α reductase inhibition as shown by lack of effects on mu ltiple androgen-dependent tissue weights.

B. AGENCY COMMENTS: One positive control (TP only) rat died after the first day of dosing due to a gavage error. All other animal s survived until scheduled termination. There were no treatment-related c linical signs of toxicity.

In the androgen agonist a ssay, body weights for the 50 and 250 mg/kg/day treatment groups were comparable to vehicle controls th roughout the duration of the assay. At 250 mg/kg/day, overall (Days 1-11) body weight gains were decreased (p ≤ 0.05) by 40%. Animals dosed with TP had increased (p ≤ 0.05) body weights on Day 11 ( ↑ 10%) and increased overall body weight gains ( ↑ 69%) compared to controls. Animals in the positive control (TP) group had increases (p ≤ 0.05) in accessory sex organ weights as follows: 1578% in seminal vesicles; 1542% in ventral prostate; 259% in LABC; 550% in Cowper’s gland; and 70% in glans penis. The maxi mum guideline-specified %CV was slightly exceeded for the seminal vesicle %CV in th e 250 mg/kg/day EPTC group (43% vs. 40%). All other %CV values were less than the maximum permissible values.

In the anti-androgen assay, body weights in the 50 and 150 mg/kg/day groups were comparable to controls throughout the durati on of the assay. At 250 mg/kg/day, overall body weight gains were decreased (p ≤ 0.05) by 34%. For the positive control group (TP + FT), overall body weight gains were decrease d (NS) by 20%. At 50 mg/kg/day, the weights of seminal vesicles were decreased by 23% (p ≤ 0.05). The LABC weights were decreased by 10% (p ≤ 0.05) at 150 mg/kg/day and 16% (p ≤ 0.05) at 250 mg/kg/day. Animals dosed with TP + FT (positive control) had decreases (p ≤ 0.05) in accessory sex organ weights as follows: 88% in seminal vesicles; 93% in ventral prostate; 55% in LABC; and 65% inIn vivo Hershberger Assay (2011)/ Page 13 of 13 EPTC/041401 OCSPP 890.1400/ OECD 441 

### Cowper’s gland. The maximum guideline-spec ified %CV was slightly exceeded for the glans penis in the 250 mg/ kg/day (19% vs. 17%) group. All other %CV values were less than the maximum permissible values.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. EPTC was negative for a ndrogenicity and anti-androgenicity in the Hershberger assay.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 In the androgen agonist assay, the maxi mum guideline-specified %CV was slightly exceeded for the seminal vesicle in the 250 mg/kg/day EPTC group (43% vs. 40%). All other %CV values were less than the maximum recommended values.

 In the anti-androgen assay, the maximum recommended %CV value was slightly exceeded for the glans penis in the 250 mg /kg/day EPTC group (19% vs. 17%) group. All other %CV values were less than the maximum recommended values.

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-05-2012 (MRID 48827603)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 7/23/2012
Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 8/13/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 8/14/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 8/14/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).EPTC I 041401 

### Primary Reviewer: Chester Rodriguez, Ph.D.

__ _ 

### Health Effects Division Secondary Reviewer: Elizabeth Mendez, Ph

. D. 

### Health Effects Division Female Pubertal Assay (2012) I Page I of 16 OCSPP 890.1450/ OECD None

# SignaM~/~~ Date:

. Signature:

# C~ Date:**~** 

### j

-

# Template~~ 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Female Pubertal Assay ; OCSPP 890.1450; OECD None.



### PC CODE: 041401 

### DP BARCODE: D402841

TXR #: 

### 0051762 

### CAS #: 

### 759-94-4



### TEST MATERIAL (PURITY): EPTC ( > 99.9% a.i.)



### SYNONYMS: S-ethyl N.N-dipropylcarbamothioate



### CITATION: Zorrilla, L. (2012) Pubertal Development and Thyroid Function with EPTC in Juvenile/Peripubertal CRL: CD (SD) IGS Male and Female Rats

. Integrated Laboratory Systems
, Inc.
, Durham
, NC. Laboratory Study No.: C201-300
, May 1
, 2012. MRID 48827603. Unpublished.



### SPONSOR: Gowan Company, P.O

. Box 5569
, Yuma
, 

### A2

TEST ORDER#: EDSP-041401-64



### EXECUTIVE SUMMARY: 

### In a Female Pubertal Assay (MRID # 48827603)
, 16 female Sprague-Dawley (Crl:CD ® (SD) IGS) rats / dose group were treated daily via oral gavage with EPTC (99.9% a.i.
, Lot #: Pl 105096) in corn oil at doses of 0, 137.5
, or 275 mg / kg / day from post-natal day (PND) 22 to 42 or 43

. Animals were examined for vaginal opening (VO) daily beginning on PND 22
, and age and weight at day of attainment were recorded. Following sacrifice on PND 42 or 43, blood was collected for clinical chemistry and hormone analyses. Total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using radioimmunoassays. Adrenal
, liver, pituitary
, thyroid
, and urogenital organs were weighed and examined macroscopically. The uterus , ovary , thyroid
, and kidney were examined microscopically

.

No adverse, treatment-related related effects were observed on survival
, clinical signs
, organ weights
, clinical chemistry
, gross pathology
, uterine or renal histology.

The following decreases (p :S 0.05) were noted in growth between the EPTC

- treated groups and the controls(% difference): (i) 

### unadjusted and adjusted body weight at VO at 275 mg / kg / day (12% ; adjusted means not reported); (ii) adjusted final body weight at 137.5 mg / kg / day (7%) and unadjusted and adjusted final body weight at 275 mg / kg / day (8% each); and (iii) unadjusted and adjusted overall bodyweight gain at 137.5 (11% each) and 275 mg / kg / day (12-13%).Female Pubertal Assay (2012) / Page 2 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None


### The relative adrenal we ight was increased (p ≤ 0.001) by 22% at 275 mg/kg/day; however, absolute and adjusted adrenal weights in the trea ted groups were similar to controls. Therefore, this effect was not considered adverse. All other organ weights in the treated groups were similar to controls.

Ages at initial and complete VO in the treated groups were similar to control, and VO occurred in all animals. No treatment-related effect was observed on the mean age of first vaginal estrus, mean cycle length, % cycling, % regularly cycling, and the cycle status at necropsy. The number of antral follicles in the ovaries was increased (p ≤ 0.05) by 145% at 275 mg/kg/day (4.9 vs. 2 in controls), and a similar trend (not significant, NS ) was noted in the number of antral follicles at 137.5 mg/kg/day (3.7).

Compared to the control, there was a significant decrease (p<0.05) in mean colloid area in the thyroid and a significant increase in mean follicular cell height at 275 mg/kg/day; similar trends (NS) in colloid area and follicular cell height were also noted at 137.5 mg/kg/day. The number of animals with colloid areas at Grade 4 decreased from 13/15 in the control to 10/16 and 5/16 at 137.5 and 275 mg/kg/day, respectively. Animals with follicular cell heights at Grade 3 increased from 2/15 in the control, to 6/16 and 10/16 at 137.5 and 275 mg/kg/day, respectively. However, the biological significance of th ese thyroid changes is unclear as there were no associated changes in serum T 4 

### and TSH levels or thyroid weight.

The dose level tested were determined to be ad equate based on the decrease in body weight and body weight gain at 275 mg/kg/day.

The assay satisfies the EDSP Tier 1 Test Order require ments for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Female Pubertal Assay (2012) / Page 3 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratories Systems, Inc.
Location: Durham, NC Study Directors: Leah Zorilla, Ph.D. Other Personnel: Susan Borghoff, Ph.D., DABT; study toxicologist Dipak, K. Giri, DVM PhD, DACVP Seena Polivy; principal investigator for hormone and clinical chemistry analyses Study Period: July 21, 2011 through May 1, 2012



### 2. Test Substance: EPTC; S -ethyl N,N -dipropylcarbamothioate Description: Off white solid Source: Gowan Company Lot #: P1105096 (expires February 2, 2015) Purity: >99.9%. Stability: Report stated that the compound is stable in the vehicle at 1-10°C for up to 25 days CAS #: 759-94-4 Structure:

N S O CH

3

CH 3 CH 3



### 3. Vehicle: Corn oil

4. Test Animals: Species: Rat (females) Strain: Sprague-Dawley (Crl:CD ® (SD) IGS) Age/ Weight at Study Initiation: PND 22 / 48.0-67.1 g
Source: Charles River Laboratories International, Inc. (Raleigh, NC) Housing: Parental dams were individual ly housed in polycarbonate cages with micro-isolator tops and absorbent, heat-treated, hardwood bedding; each dam was housed with her litter until weaning on PND 21. After weaning, up to 2 females were housed per cage. Diet: Teklad Global 16% Protein Rodent Di et (Teklad Diets, Madison, WI), ad libitum . It was stated that the total genistein equivalents of genistein plus daidzein was determined to be 8.0-8.6 μ g/g of feed. Water: Reverse osmosis-purified tap water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 17-23°C 33-77% Not reported 14 h light / 10 h dark



### B. STUDY DESIGN

1. In-Life Dates: Start: August 26, 2011 End: September 17, 2011

2. Mating: Time-mated pregnant nulliparous dams (age not reported) were received from the supplier on gestation day 8 and we re allowed to deliver naturall y and rear their pups to PND 21. Between postnatal days (PND) 3 and 5, litters with the same date of birth were standardized to 8 pups with equa l numbers of males and females.Female Pubertal Assay (2012) / Page 4 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None



### 3. Animal Assignment: Following weaning on PND 21, animals were assigned to study using a procedure that stratifies animals across groups by body weight such that mean body weight of each group was not st atistically different from any other group using analysis of variance. Care was taken to avoid placing li ttermates in the same dose group. The test groups are provided in Table 1.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Females
Control 0 16
Low 137.5 16 High 275 16 a Data were obtained from Table 1 on page 15 of the study report.



### 4. Dose Selection Rationale: The doses were selected base d on the results from a range finding study 1 

### and other previously conducted stud ies in female rats with orally administered EPTC. In the range finding study, juvenile female rats were given EPTC at dose levels of 0, 200, 400, or 600 mg/kg/day for 14 days beginning on PND 22. Mortality occurred at 600 mg/kg/day, and the mean body weights on the last day of dosing (PND 35) for the 400 and 200 mg/kg/day groups were 84% and 103%, respectively, of the vehicle control. Maternal mortality was observed on a previous developmental toxicity study at doses >300 mg/kg/day EPTC, albeit using older animals. Therefore, considering the young animal model used in this assay, and the num ber of days the animals are dosed, a high dose of 275 mg/kg/day was chosen for the female pubertal assay. The second dose (137.5 mg/kg/day) was one-half the high dose.

5. Dose Preparation and Analysis: EPTC dose formulations were prepared at ILS in corn oil twice (August 18, 2011 and September 6, 2011) duri ng the study at con centrations of 27.5 and 55 mg/mL. EPTC and corn oil were adju sted based on their density, weighed, and were mixed on a stir plate until dissolved. All EPTC formulation concentrations were solutions. Formulations were dispensed into vial s to be used daily during the study.

Samples of each dose formulation were collected from the top, middle, and bottom of the formulations and sent to Smithers Viscient (W areham, MA) for analysis. Smithers Viscient analyzed, in duplicate, samples received fo r concentration and homogeneity. The Report stated that the compound is stable in the vehi cle at 1-10°C for up to 25 days on the basis of previous study 2 

### ; however, data were not presented.

1 Zorrilla, L. (2012). Range Finder Study for the In Vivo Pubertal Assays for EPTC. Unpublished study report

prepared by ILS, Inc. Study No. C201-500.
2 Dix, R. (2011). Storage Stability of EPTC in Corn Oil. Unpublished study report prepared by Smithers Viscient,

LLC. Study No. 12791-6162.Female Pubertal Assay (2012) / Page 5 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None



### Results

Homogeneity (CV): 0.564-3.34%

Stability (% of time zero): Not reported

Concentration (% of nominal): 97.2-103%
The analytical data indicate d that the mixing procedure wa s adequate and the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: Dose formulations were administered once daily by oral gavage in a dose volume of 5 mL/kg body weight. The dose formulations were administered for 21 or 22 consecutive days from PND 22-42/43. Do sing occurred 24-hours (± 2 hours) from the previous dose. Dose volume was adjusted daily based on individual animal body weight. The daily dosing sequence was stratified across all dose groups; 1 animal from each group and then repeated until all animals were dosed.

7. Statistics: Descriptive statistics (mean, standard deviation, co efficient of variance, and sample size) were calculated using Microsof t Excel 2003/2007 (Redmond, WA). Data sets listed below were analyzed using Statistical Analysis System version 9.2 (SAS Institute, Cary, NC). Studentized residual plots were used to detect possi ble outliers in the data and Levene’s test was used to assess homogeneity of variance. Heterogeneous data were transformed (logarithm, square root, and multiplicative inverse) and if still heterogeneous analyzed using the non-parametric Kruskal-Wallis and Dunn’s test.

Classification Procedure Initial body weights Final body weight (use last day all body weights collected) Cumulative body weight gain (use last day all body weights collected) Age and body weight at VO Age at first estrus Cycle length Ovarian counts (follicles, cysts, corpora lutea) Homogeneous data sets:One-way analysis of variance (ANOVA) followed by pairwise comparisons performed using Dunnett’s two-tailed t-tests

Body weight Organ weight Tests for linear trend were performed and reported when significant (p<0.05) for endpoints that did not show any significant pair-wise comparisons. Tissue weights Relative tissue weights (liv er, kidneys, pituitary, and adrenals) Hormone levels Clinical chemistry levels Two-way ANOVA with treatment and necropsy day (if >1 day) as main effects. Pair-wise comparisons were performed using Dunnett’s two-tailed t-tests.

Final body weight (use last day all body weights collected) Final body weight gain (use last day all body weights collected) Age and body weight at VO Tissue weights Analysis of covariance (ANCOVA) with PND 21 body weight (weight on day of allo cation) as the covariable. Pair-wise comparisons were performed using Dunnett’s two-tailed t-tests.

Cycling status Chi-square analysisFemale Pubertal Assay (2012) / Page 6 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None

Classification Procedure Percent of animals cycling regularly Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test, and when statistically significant, followed by Kruskal-Wallis and Dunn's test.



### Significance was reported as exact p-values, ex cept no p-value was reported for Dunn's Test (p<0.05). The statistical analyses were considered appropriate.

C. METHODS

1. Mortality and Clin ical Examinations: Animals were observed twice daily for mortality and moribundity on weekdays and once daily on weekends and holidays. Rats were observed prior to allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia.

2. Body Weight: Body weights were recorded weekly following birth unti l allocation of animals to study groups. Then body weights were recorded daily prior to dosing, and final body weight was recorded just prior to euthanasia.

3. Food Consumption: Food consumption was not measured.

4. Vaginal Opening (VO): Beginning on PND 22, females were examined daily (approximately the same time each day) followi ng dosing for VO. VO was recorded as: VO not initiated, appearance of a pin hole, vaginal thread, or complete VO.

5. Estrous Cyclicity: Beginning on the day of complete VO through euthanasia, vaginal smears were prepared daily after dose admini stration (approximately the same time each day), evaluated, and classified as diestrus, proestrus, or estrus . Metestrus was classified as diestrus. The overall cycling pa ttern for each female was charact erized as regular, irregular, or not cycling. Regular cycling was defined as having recurring 4- to 5-day cycles. Animals were considered not to be cycling if there was ≥ 3 consecutive days of estrus or ≥ 5 consecutive days of diestrus.

6. Sacrifice and Pathology: Animals were moved to the necropsy holding room at least 2 hours before euthanasia. Animals were hum anely euthanized by decapitation 2-5 hours post-dose, in the same order as they were dosed. After euthanasia and prior to tissue collection, trunk blood was collected, and seru m was obtained. All serum samples were stored at or below

− 70°C. Serum samples were shippe d on dry ice to AniLytics Inc. (Gaithersburg, MD) for analysis.

a. Hormone Analysis: Total T 4 

### and TSH were measured in serum using radioimmunoassays. Samples were assayed in duplicate and in conjunction with multiple quality control samples.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHERFemale Pubertal Assay (2012) / Page 7 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None

X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus Total cholesterol X Potassium Globulins
X Sodium Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Albumin /globulin ratio (calculated) Lactic acid dehydrogenase (LDH) Bile acids X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) X Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on guideline 890.1450.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed and grossly observed fr om all study animals. The (XX) organs, in addition, were subjected to histological examination.

UROGENITAL OTHER XX Ovaries (paired, without oviducts)*+ XX Thyroid a * + XX Uterus*+ X Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Pituitary* a Thyroid was weighed post-fixation. * Weights required base d on guideline 890.1450. + Histopathological examination re quired based on guideline 890.1450.



### Uterus (without fluid) left ovary, left kidney, and thyroid (with attach ed trachea) were fixed in 10% neutral buffered formalin for at leas t 24-hours. Following fi xation, the thyroid was dissected free of the trachea and weighed. Af ter fixation, tissues were transferred to 70% histology grade alcohol, routinel y processed into paraffin blocks , sectioned, and stained with hematoxylin and eosin. Histologically-processe d tissues (uterus without fluid, left ovary, thyroid, and left kidney) from all animals were microscopica lly evaluated. Evaluation of each ovarian section included: follicular de velopment (including presence/absence of tertiary/antral follicles, presence/absence of corpora lutea, change s in corpus luteum development, and changes in number of both primary and atretic follicles) in addition to any abnormalities/lesions, such as ovarian atrophy. Tw o sections of each of the 2 lobes of the thyroid were examined and evaluated for follicular cell height and colloid area. A 5-point grading scale (1 = shortest/smallest; 5 = tall est/largest) was used, and the thyroids were evaluated relative to photomicrogr aphs supplied by the U.S. EPA.

II. RESULTSFemale Pubertal Assay (2012) / Page 8 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None



### A. MORTALITY: One rat (No. 14) administered vehicl e control (Group 1) was found dead on PND 42 after receiving its daily dose. Upon necropsy, the anim al had oil droplets in the lung lavage, and this death was attributed to a gavage error. All rema ining animals survived to study termination.

B. CLINICAL SIGNS OF TOXICITY: No treatment-related clin ical signs were observed. Rough coat was noted on at least 1 day in 3 control animals, 6 animals at 137.5 mg/kg/day, and 4 animals at 275 mg/kg/day. However, this finding was considered incidental as these animals were normal all other days of the st udy. All other rats in these groups appeared normal throughout the study.

C. GENERAL GROWTH AND VAGINAL OPENING: Body weights, body weight gains, age of attainment of VO, we ight at VO, and proportion/incidence of unopened are presented in Table 2. The following decreases (p ≤ 0.05) were noted between the EPTC-treated groups and the controls (% difference): (i) unadju sted and adjusted body weight at VO at 275 mg/kg/day (12%; adjusted means not reported) ; (ii) adjusted final body weight at 137.5 mg/kg/day (7%) and unadjusted and adjusted final body weight at 275 mg/kg/day (8% each); and (iii) unadjusted and adjusted ove rall bodyweight gain at 137.5 (11% each) and 275 mg/kg/day (12-13%). Initial body weights an d ages at initial and complete VO in the treated groups were similar to cont rol. VO occurred in all animals.

The Guideline performance criteria were me t for age and weight at VO and final body weight.Female Pubertal Assay (2012) / Page 9 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None

TABLE 2. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Dose levels

Vehicle Control EPTC 137.5 mg/kg/day EPTC 275 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 22; g) U 16 57.2 4.1 7.1 16 57.5 4.4 7.6 16 57.6 3.8 6.6 A 16 57.4 --- --- 16 57.4 --- --- 16 57.5 --- --- Body weight at VO (g) U 16 114.9 8.7 7.6 16 111.8 13.0 11.6 16 100.6*** ( ↓ 12) 6.9 6.8

A 16 NR --- --- 16 NR --- --- 16 NR*** --- ---

Final body weight (g) U 15 169.7 14.0 8.3 16 157.8 11.7 7.4 16 155.9* ( ↓ 8) 18.1 11.6

A 15 170.0 --- --- 16 157.7* ( ↓ 7) --- --- 16 155.8** ( ↓ 8) --- --- Final body weight (% of control) U 15 --- --- --- 16 93.0 --- --- 16 91.9 --- --- A 15 --- --- --- 16 92.8 --- --- 16 91.6 --- ---

Body weight gain (final – initial; g) U 15 112.3 11.9 10.6 16 100.4* ( ↓ 11) 9.9 9.9 16 98.4** ( ↓ 12) 15.6 15.9

A 15 112.5 --- --- 16 100.3* ( ↓ 11) --- --- 16 98.3** ( ↓ 13) --- --- Age at initial VO (PND) U 16 32.5 1.6 4.9 16 32.6 2.1 6.4 16 31.4 1.0 3.3
A 16 32.5 --- --- 16 32.6 --- --- 16 31.4 --- --- Age at complete VO (PND) U 16 32.5 1.6 4.9 16 32.6 2.1 6.4 16 31.4 1.0 3.3 A 16 32.5 --- --- 16 32.6 --- --- 16 31.4 --- --- Proportion unopened (#/N) 0/16 0/16 0/16 a Data were obtained from Table 5 on page 26 of the study report. Percent differences from controls (calculated by reviewers) are included in parentheses. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 N Number of animals examined SD Standard Deviation SE Standard Error; used for means adjusted for body weight on PND 21 CV Coefficient of Variation NA Not applicable NR No value was reported * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01. *** Significantly different from controls at p ≤ 0.001.



### D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. The relative adrenal weight was increased (p ≤ 0.001) by 22% at 275 mg/kg/day compared to controls. All other organ weights in the treated groups were similar to controls.

The unadjusted values for all organ weights in the control group were within the acceptable range of the performance criteria provided in the Guideline (8 90.1450), with the exception of the mean adrenal weight of 38.0 mg (acceptable range: 38.34-48.84 mg).Female Pubertal Assay (2012) / Page 10 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control 137.5 mg/kg/da y EPTC 275 mg/kg/day EPTC

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 15 8.05 0.95 11.8 16 7.41 0.80 10.8 16 7.72 1.04 13.5
A 15 8.07 --- --- 16 7.41 --- --- 16 7.71 --- ---
R 15 4.74 0.34 7.3 16 4.69 0.27 5.7 16 4.94 0.23 4.6

Kidneys (g) U 15 1.3 0.08 6.3 16 1.26 0.14 11 16 1.26 0.14 10.8
A 15 1.3 --- --- 16 1.26 --- --- 16 1.26 --- ---
R 15 0.77 0.06 7.4 16 0.8 0.06 7 16 0.81 0.06 7.7

Pituitary (mg) U 15 9.4 1.0 10.3 16 8.7 1.2 13.3 16 8.8 1.1 13.0
A 15 9.4 --- --- 16 8.7 --- --- 16 8.8 --- ---
R 15 5.6 0.9 15.2 16 5.5 0.7 12.3 16 5.7 0.8 13.9

Adrenals (mg) U 15 38.0 4.9 13 16 39 5.3 13.5 16 42.6 6.7 15.7
A 15 38.1 --- --- 16 39 --- --- 16 42.6 --- ---

R 15 22.5 2.6 11.6 16 24.8 3.0 12.2 16 27.4*** ( ↑ 22) 3.5 12.8

Ovaries (mg) U 15 74.4 10.1 13.6 16 74.5 12.5 16.8 16 80.7 16.7 20.7
A 15 74.6 --- --- 16 74.4 --- --- 16 80.6 --- ---
Uterus, wet (mg) U 15 342.4 127.1 37.1 16 325 137.2 42.2 15 343.8 130.4 37.9
A 15 342.4 --- --- 16 325 --- --- 15 343.8 --- --- Uterus, blotted (mg) U 15 294.4 75.6 25.7 16 268.6 75.1 28.0 16 284.6 65.9 23.2

A 15 294.4 --- --- 16 268.6 --- --- 16 284.6 --- ---
Thyroid (mg) U 15 13.6 2.3 16.8 16 13.4 2.8 20.6 16 13.4 2.0 14.7
A 15 13.6 --- --- 16 13.4 --- --- 16 13.4 --- --- a Data were obtained from Table 6 on page 28 of the study repor t. Percent difference from controls (calculated by reviewers) is presented pa renthetically. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 R Organ-to-body weight ratio (relative to body weight) SD Standard Deviation CV Coefficient of Variation *** Significantly different fro m controls at p<0.001.



### E. ESTROUS CYCLICITY: Estrous cycle data are provi ded in Table 4. No treatmentrelated effect was observed on the mean age of first vaginal estrus, mean cycle length, % cycling, % regularly cycling, and the cycle status at necropsy.Female Pubertal Assay (2012) / Page 11 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None

TABLE 4. Estrous Cyclicity a

Treatment Groups N Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) Cycling (%) Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling Vehicle 16 32.8 4.6 100 100 9 3 3 0 EPTC (137.5 mg/kg/day) 16 33.4 4.8 100 87.5 (n=14) 9 3 3 0

EPTC (275 mg/kg/day) 16 31.9 4.6 100 100 9 2 4 0 a Data were obtained from Table 6 on page 28 and Table 7 on page 29 of the study report. No differences (p ≤ 0.05) from the controls were detected in the treated groups.



### F. HORMONE LEVELS AND CLINICAL CHEMISTRY: Clinical chemistry and hormone levels are presented in Table 5. No rmal ranges for these values were provided. Serum T 4 

### and TSH levels in the treated groups were similar to the controls. The following clinical chemistry parameters differed (p ≤ 0.01) in the treated groups compared to controls: (i) sodium increased by 2-4% at both doses; (ii) chloride increased by 3-4% at both doses; (iii) calcium decreased by 3% at 275 mg/kg/day; (i v) blood urea nitrogen decreased by 18% at 275 mg/kg/day; (v) SDH increased by 32% at 275 mg/kg/day; and (v i) albumin decreased by 7% at 275 mg/kg/day. Thes e differences were minor and, with the exception of sodium and blood urea nitrogen, were within the reported normal range.

Serum T 4 

### mean and CV in the control group were within the acceptable range of the performance criteria provid ed in the Guideline (890.1450).

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control 137.5 mg/kg/da y EPTC 275 mg/kg/day EPTC

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Hormones
Serum T 4 (μg/dL) 15 3.41 0.96 28.2 16 2.93 0. 41 14.0 16 3.46 0.98 28.2

Serum TSH (ng/mL) 15 2.21 0.73 33.2 16 2.08 1. 14 55.0 16 2.24 1.13 50.8
Clinical Chemistry Sodium (mEq/L) 15 135 2 1.4 16 138*** ( ↑ 2) 2 1.6 16 140*** ( ↑ 4) 2 1.2

Potassium (mEq/L) 15 7.9 0.6 7.3 16 8 1 6.6 16 7.9 0.5 5.9

Chloride (mEq/L) 15 101 2 1.6 16 104*** ( ↑ 3) 1 1.4 16 105*** ( ↑ 4) 1 1.4

Calcium (mg/dL) 15 10.2 0.3 2.7 16 10.1 0.2 2.4 16 9.9** ( ↓ 3) 0.4 3.7
Phosphorus (mg/dL) 15 9.2 0.7 8 16 9.3 0.5 5.5 16 9.5 0.9 9.1
AST (U/L) 15 293 74 25.2 16 282 38 13.6 16 273 45 16.3 ALT (U/L) 15 110 21 19.1 16 104 12 11.3 16 104 17 16 GGT (U/L) 15 1 1 186 16 1 1 174 16 0 0 215 ALP (U/L) 15 365 92 25.2 16 331 56 16.8 16 323 84 26.1Female Pubertal Assay (2012) / Page 12 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control 137.5 mg/kg/da y EPTC 275 mg/kg/day EPTC

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
BUN (mg/dL) 15 11 2 19.1 16 10 2 16.5 16 9*** ( ↓ 18) 2 18.2

Creatinine (mg/dL) 15 0.4 0 13.3 16 0.4 0.1 14.4 16 0.3 0.1 14.9

Total Bilirubin (mg/dL) 15 0.01 0.03 387 16 0 0 --- 16 0.03 0.04 179

SDH (U/L) 15 19 5 25.9 16 23 5 20.4 16 25** ( ↑ 32) 5 20.6

Total Protein (g/dL) 15 5.7 0.2 4.2 16 5.8 0.3 5.2 16 5.6 0.2 3.5

Albumin (g/dL) 15 4.3 0.2 4.8 16 4.3 0.2 4.9 16 4** ( ↓ 7) 0.2 5.8
a Data were obtained from Table 8 on page 29 and Table 9 on page 31 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically. SD Standard Deviation CV Coefficient of Variation ** Significantly different fro m controls at p<0.01. *** Significantly different fro m controls at p<0.001.



### G. GROSS PATHOLOGY: There were no macroscopic findings observed in the uterus, ovary, thyroid gland, or kidney reported in any animal.

H. HISTOPATHOLOGY: The incidences of histopathologi cal findings of the thyroid gland are presented below in Table 6; follicular cell shape was not reported. A trend toward decreasing colloid area and increasing follicular cell height was noted in the thyroids of EPTC-treated animals. The number of animals with colloid areas at Grade 4 decreased from 13/15 in the control to 10/16 and 5/16 at 137.5 and 275 mg /kg/day, respectively. Animals with follicular cell heights at Grade 3 increased from 2/15 in the control, to 6/16 and 10/16 at 137.5 and 275 mg/kg/day, respectively. The changes in colloid area and cell height attained significance (p<0.05) at the 275 mg/kg/day dose.Female Pubertal Assay (2012) / Page 13 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None

TABLE 6. Incidence of Histopathol ogical Lesions of the Thyroid Gland a

Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height (Increase) Severity b Incidence Grade b Incidence O E O E

Vehicle Control 1 0 15 1 0 15 2 0 15 2 13 15 3 2 15 3 2 15 4 13 15 4 0 15 5 0 15 5 0 15

137.5 mg/kg/day EPTC 1 0 16 1 0 16 2 0 16 2 10 16 3 6 16 3 6 16 4 10 16 4 0 16 5 0 16 5 0 16

275 mg/kg/day EPTC* 1 0 16 1 0 16 2 1 16 2 5 16 3 10 16 3 10 16 4 5 16 4 1 16 5 0 16 5 0 16 a Data were obtained from Table 12 on page 34 of the study report. b A 5-point grading scale (1 = shortest/s mallest; 5 = tallest/largest) was used. O No. Observed E No. Examined * Significantly different fro m controls at p<0.05.



### The incidences of histopathological findings of the ovaries are presented in Table 7. The number of antral follicles in the ovaries was increased (p ≤ 0.05) by 145% at 275 mg/kg/day (4.9 vs. 2 in controls), and a si milar trend (NS) was noted in th e number of antral follicles at 137.5 mg/kg/day (3.7). The inciden ces of other ovarian findings we re similar to controls.

TABLE 7. Number of Ovarian Follicles, Follicular Cysts, and Corpora Lutea a

Parameter Evaluated Vehicle Control 137.5 mg/kg/da y EPTC 275 mg/kg/day EPTC

N b Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Small follicles 14 35.1 34.4 98 16 38.7 26.3 68 16 27.3 24.3 89 Medium follicles 14 13.5 9 67 16 18.5 8.2 44 16 13.6 9.1 67

Antral follicles 14 2 2 100 16 3.7 1.6 43 16 4.9* ( ↑ 145) 3.9 80

Atretic follicles 14 7.5 6.2 83 16 10.3 6.4 62 16 8.2 6.5 79 Follicular cysts 14 0.07 0.27 386 16 0 0 - 16 0 0 - Corpora lutea 14 6.7 2.2 33 16 8.6 2.8 33 16 8.4 2.8 33 a Data were obtained from Table 11 on page 33 of the stud y report. Percent difference from controls (calculated by reviewers) is presented parenthetically. b One animal died prior to necropsy, and another animal had predominantly stromal tissue and was not included. SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05.Female Pubertal Assay (2012) / Page 14 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None 

### The incidences of histopathologi cal findings of the uterus and ki dneys are presented in Table 8, and no treatment-related findings were observed.

TABLE 8. Histopathological Findings in the Uterus and Kidneys

Parameter Vehicle Control EPTC 137.5 mg/kg/day EPTC 275 mg/kg/day Uterus Dilatation Minimal 2 6 7 Mild 3 0 0 Moderate 0 0 1 Eosinophilic materi al, Minimal 0 1 1 Inflammatory cells/debris Minimal 0 1 1 Mild 0 1 0 Endometrial folding Minimal 8 8 8 Mild 0 0 1 Endometrium: inflammatory cells Minimal 7 1 0 Mild 0 1 1 Kidney Nephropathy, Minimal 8 5 9 Cysts present 2 0 2 a Data (n=16, except n=15 for contro ls) were obtained from Appendix 2 on page 128 and Appendix 12 on page 138 of the study report.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Oral administration of EPTC up to 275 mg/kg/day (approaching the MTD) to intact juvenile/peripubertal female rats for at least 21 days, did not appear to affect pubert al development or thyroid function.

B. AGENCY COMMENTS: No adverse, treatment-relate d related effects were observed on survival, clinical signs, organ weights, clinical chemistry, gr oss pathology, or uterine or renal histology. One control rat was found dead on PND 42 due to gavage error.

The following decreases were noted in growth between the treated groups and the controls: (i) unadjusted and adjusted body weight at VO at 275 mg/kg/day; (ii) adjusted final body weight at 137.5 mg/kg/day and unadjusted and adjusted final body weight at 275 mg/kg/day; and (iii) unadjusted and adju sted overall bodyweight gain at 137.5 and 275 mg/kg/day.

The relative adrenal weight wa s increased at 275 mg/kg/day; however, the absolute and adjusted adrenal weights in the treated groups were similar to contro ls. Therefore, this effect was not considered adverse. All ot her organ weights in th e treated groups were similar to controls.

The following clinical chemistry parameters di ffered in the treated groups compared to controls: (i) sodium increased at both doses; (ii) chloride increased at both doses; (iii) calcium decreased at 275 mg/ kg/day; (iv) blood urea nitrogen decreased at 275 mg/kg/day;Female Pubertal Assay (2012) / Page 15 of 16 EPTC / 041401 OCSPP 890.1450/ OECD None 

### (v) SDH increased at 275 mg/kg/day; and (vi) albumin decreased at 275 mg/kg/day. These differences were minor and, with the excepti on of sodium and blood urea nitrogen, were within the reporte d normal range.

Ages at initial and complete VO in the treated groups were similar to control, and VO occurred in all animals. No treatment-related effect was observed on the mean age of first vaginal estrus, mean cycle le ngth, % cycling, % regularly cyc ling, and the cycle status at necropsy. Histological examination of the ovari es indicated a trend to ward increases in the number of antral follicles of EPTC-treated rats, which attained significance (p ≤ 0.05) at 275 mg/kg/day.

Compared to the control, there was a significan t decrease (p<0.05) in mean colloid area in the thyroid and a significant increase in mean follicular cell height at 275 mg/kg/day; similar trends (NS) in colloid area and follicular cell height were also noted at 137.5 mg/kg/day. However, the biological signifi cance of these thyroid changes is unclear as there were no associated changes in serum T 4 

### and TSH levels or thyroid weight.

Based on the decrease in body weight and body we ight gain, the high dose was considered adequate.

C. STUDY DEFICIENCIES: The following minor deficienci es were noted that are not considered to have had an adverse impact on th e results, interpretation or conclusions of this study:

 All performance criteria pr ovided in the Guideline ( 890.1450) were met, with the exception of the mean adrenal weight of 38.0 mg (acceptable range: 38.34-48.84 mg).

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-05-2012 (MRID 48827603)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 7/23/2012
Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 8/13/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 8/14/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 8/14/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).EPTC I 041401 

### Primary Reviewer: Chester Rodriguez, Ph.D. Health Effects Division Secondary Reviewer: John 

### Liccione, Ph.D

. Health Effects Division Male Pubertal Assay (2012) I Page I of 15 CS P 890.1500/ OECD None



### DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay ; OCSPP 890.1500 ; OECD None

.



### PC CODE: 041401 

### DP BARCODE: D40284 l

TXR #: 

### 0051762 

### CAS #: 759-94-4



### TEST MATERIAL (PURITY): EPTC ( > 99

. 9% a.i

. )



### SYNONYMS: S-ethyl N,N -dipropylcarbamothioate



### CITATION: Zorrilla
, L. 

### (2012) Pubertal Development and Thyroid Function with EPTC in Juvenile/Peripubertal CRL: CD (SD) IGS Female and Male Rats. Integrated Laboratory Systems
, Inc.
, Durham, NC. Laboratory Study No.: C201-300
, May I
, 2012

. MRID 48827603. Unpublished

.



### SPONSOR: Gowan Company
, P.O

. Box 5569
, Yuma
, AZ



### TEST ORDER#: EDSP-041401-64



### EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48827603)
, 16 male SpragueDawley (Crl:CD ® (SD) IGS) rats / dose group were treated daily via oral gavage with EPTC (99.9% a.i., Lot#: Pl 105096) in com oil at doses of 0
, 62.5
, or 125 mg / kg / day from post-natal day (PND) 23 to 53 or 54

. Animals were examined for preputial separation (PPS) daily beginning on PND 30
, and age and body weight at day of attainment was recorded. Following sacrifice on PND 53 or 54
, blood was collected for hormone assays and clinical chemistry analyses. Total serum testosterone , thyroxine (T 4 )
, and thyroid stimulating hormone (TSH) levels were analyzed using radioimmunoassays

. Thyroid
, liver
, adrenals
, pituitary
, and urogenital organs were weighed and examined macroscopically. The thyroid
, the left kidney
, epididymides
, and testis were examined microscopically.

No treatment-related related effects were observed on survival, clinical signs
, body weights, final body weight
, body weight gains, organ weights
, clinical chemistry
, gross pathology
, or renal histolog y

.

No adverse
, treatment-related related effects were observed on body weight or age at PPS
, age at partial PPS
, proportion/incidence of unseparated preputia
, androgen-dependent tissue organ weights
, or serum testosterone and thyroid hormone levels. The colloid area and follicular cell height of the thyroid in the treated groups were similar to the control group. The incidences ofMale Pubertal Assay (2012) / Page 2 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None 

### histopathological findings of the testes and epididymides in the treated groups were similar to controls.

The LABC weights showed a linear trend of decreased (p ≤ 0.05) weight with increasing dose; however, no statistically signifi cant effect was detected betw een the treated groups and the control group. No other statistica lly significant effect or trend was detected for the sex tissues or any other organ weighed. Therefore, this linear trend was c onsidered incidental.

Although no toxicity was noted at the high dose (125 mg/kg/day) and terminal body weight was only decreased by 5% (NS), relativ e to the control, the high dose is considered adequate for purposes of this assay based data from on a ra nge-finding study using juvenile males in which dosing at 200 and 400 mg/kg/day re sulted in body weight decreases of 14 and 15%, respectively

The assay satisfies the EDSP Tier 1 Test Order requirement s for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Male Pubertal Assay (2012) / Page 3 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratories Systems, Inc.
Location: Durham, NC Study Directors: Leah Zorilla, Ph.D. Other Personnel: Susan Borghoff, Ph.D., DABT; study toxicologist Dipak, K. Giri, DVM PhD, DACVP Seena Polivy; principal investigator for hormone and clinical chemistry analyses Study Period: July 21, 2011 through May 1, 2012



### 2. Test Substance: EPTC; S -ethyl N,N -dipropylcarbamothioate Description: Off white solid Source: Gowan Company Lot #: P1105096 (expires February 2, 2015) Purity: >99.9%. Stability: Report stated that the compound is stable in the vehicle at 1-10°C for up to 25 days CAS #: 759-94-4 Structure:

N S O CH

3

CH 3 CH 3



### 3. Vehicle: Corn oil

4. Test Animals: Species: Rat (males) Strain: Sprague-Dawley (Crl:CD ® (SD) IGS) Age/ Weight at Study Initiation: PND 23 / 48.1-75.1 g
Source: Charles River Laboratories International, Inc. (Raleigh, NC) Housing: Parental dams were individual ly housed in polycarbonate cages with micro-isolator tops and absorbent, heat-treated, hardwood bedding; each dam was housed with her litter until weaning on PND 21. After weaning, up to 2 males were housed per cage. Diet: Teklad Global 16% Protein Rodent Di et (Teklad Diets, Madison, WI), ad libitum . It was stated that the total genistein equivalents of genistein plus daidzein was determined to be 8.0-8.6 μ g/g of feed. Water: Reverse osmosis-purified tap water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 17-23°C 33-77% Not reported 14 h light / 10 h dark



### B. STUDY DESIGN

1. In-Life Dates: Start: August 26, 2011 End: September 27, 2011

2. Mating: Time-mated pregnant nulliparous dams (age not reported) were received from the supplier on gestation day 8 and we re allowed to deliver naturall y and rear their pups to PND 21. Between PND 3 and 5, litters with the same date of birth were st andardized to 8 pups with equal numbers of males and females.Male Pubertal Assay (2012) / Page 4 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None



### 3. Animal Assignment: Following weaning on PND 21, animals were assigned to study using a procedure that stratifies animals across groups by body weight such that mean body weight of each group was not st atistically different from any other group using analysis of variance. Care was taken to avoid placing li ttermates in the same dose group. The test groups are provided in Table 1.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males
Control 0 16
Low 62.5 16 High 125 16 a Data were obtained from Table 2 on page 15 of the study report.



### 4. Dose Selection Rationale: The doses were selected base d on the results from a range finding study 1 

### and a Hershberger Assay 2 

### in juvenile male rats with orally administered EPTC. In the range finding study, rats were given EPTC at dose levels of 0, 200, 400, or 600 mg/kg/day for 14 days beginning on PND 2 2. Mortality occurred at 600 mg/kg/day, and the mean body weights on the last day of dosing (PND 35) for the 400 and 200 mg/kg/day groups were 86% and 85%, respectively, of the vehicle control. In the Hershberger Bioassay, there was a statistically significant lower body we ight gain after 10 days of administration of 250 mg /kg/day EPTC compared to ve hicle control. Therefore, considering the young animal model used in this assay, and the number of days the animals are dosed, a high dose of 125 mg/kg/day was chosen for the male pubertal assay to meet the criteria for targeting the maximum tolerated dose. The second dos e (62.5 mg/kg/day) was one-half the high dose as described by the guidelines.

5. Dose Preparation and Analysis: EPTC dose formulations were prepared at ILS in corn oil twice (August 18, 2011 and September 6, 2011) duri ng the study at con centrations of 12.5 and 25 mg/mL. EPTC and corn oil were adju sted based on their density, weighed, and were mixed on a stir plate until dissolved. All EPTC formulation concentrations were solutions. Formulations were dispensed into vial s to be used daily during the study.

Samples of each dose formulation were collected from the top, middle, and bottom of the formulations and sent to Smithers Viscient (W areham, MA) for analysis. Smithers Viscient analyzed, in duplicate, samples received fo r concentration and homogeneity. The Report stated that the compound is stable in the vehi cle at 1-10°C for up to 25 days on the basis of previous study 3



### .

1 Zorrilla, L. (2012). Range Finder Study for the In Vivo Pubertal Assays for EPTC. Unpublished study report

prepared by ILS, Inc. Study No. C201-500.
2 Zorrilla, L. (2011). The Hershberger Assay with EPTC in Castrated CRL:CD (SD) IGS Rats. Unpublished study

report prepared by ILS, Inc. Study No. C201-200.
3 Dix , R. (2011). Storage Stability of EPTC in Corn Oil. Unpublished study report prepared by Smithers Viscient,

LLC. Study No. 12791-6162.Male Pubertal Assay (2012) / Page 5 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None



### Results

Homogeneity (CV): 0.603-3.79%

Stability (% of time zero): Not reported

Concentration (% of nominal): 99.3-104%
The analytical data indicate d that the mixing procedure wa s adequate and the variation between nominal and actual dosage to the animal s was acceptable. Stability data should be submitted for independent verification.

6. Dosage Administration: Dose formulations were administered once daily by oral gavage in a dose volume of 5 mL/kg body weight. The dose formulations were administered for 31 or 32 consecutive days from PND 23-53/54. Do sing occurred 24-hours (± 2 hours) from the previous dose. Dose volume was adjusted daily based on individual animal body weight. The daily dosing sequence was stratified across all dose groups; 1 animal from each group and then repeated until all animals were dosed.

7. Statistics: Descriptive statistics (mean, standard deviation, co efficient of variance, and sample size) were calculated using Microsof t Excel 2003/2007 (Redmond, WA). Data sets listed below were analyzed using Statistical Analysis System version 9.2 (SAS Institute, Cary, NC). Studentized residual plots were used to detect possi ble outliers in the data and Levene’s test was used to assess homogeneity of variance. Heterogeneous data were transformed (logarithm, square root, and multiplicative inverse) and if still heterogeneous analyzed using the non-parametric Kruskal-Wallis and Dunn’s test.Male Pubertal Assay (2012) / Page 6 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None

Classification Procedure Initial body weights Final body weight (use last day all body weights collected) Cumulative body weight gain (use last day all body weights collected) Age and body weight at PPS Homogeneous data sets:One-way analysis of variance (ANOVA) followed by pairwise comparisons performed using Dunnett’s two-tailed t-tests

Body weight Organ weight Tests for linear trend were performed and reported when significant (p<0.05) for endpoints that did not show any significant pair-wise comparisons. Tissue weights Relative tissue weights (liv er, kidneys, pituitary, and adrenals) Hormone levels Clinical chemistry levels Two-way ANOVA with treatment and necropsy day (if >1 day) as main effects. Pair-wise comparisons were performed using Dunnett’s two-tailed t-tests.

Final body weight (use last day all body weights collected) Final body weight gain (use last day all body weights collected) Age and body weight at PPS Tissue weights Analysis of covariance (ANCOVA) with PND 21 body weight (weight on day of allo cation) as the covariable. Pair-wise comparisons were performed using Dunnett’s two-tailed t-tests.

In the instances where PPS had not occurred prior to necropsy, the last day of examination plus 1 was used as the age at PPS and terminal body weight was used for body weight at PPS. In instances when at least 1 animal in any group exhibited incomplete PPS, including partial threads for > 3 days, the day partial separation was first recorded was used in the analyses. Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test, and when statistically significant, followed by Kruskal-Wallis and Dunn's test.



### Significance was reported as exact p-values, ex cept no p-value was reported for Dunn's Test (p<0.05). The statistical analyses were considered appropriate.

C. METHODS

1. Mortality and Clin ical Examinations: Animals were observed twice daily for mortality and moribundity on weekdays and once daily on weekends and holidays. Rats were observed prior to allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia.

2. Body Weight: Body weights were recorded weekly following birth unti l allocation of animals to the study groups. Then body weight s were recorded daily prior to dosing, and final body weight was recorded just prior to euthanasia.

3. Food Consumption: Food consumption was not measured.

4. Preputial Separation (PPS): Beginning on PND 30, males were examined daily following dosing, and PPS was noted as: pr ogression not initiated, partia l separation, a persistent thread of tissue between the glan s and prepuce, or complete PPS.

5. Sacrifice and Pathology: Animals were moved to the necropsy holding room at least 2 hours before euthanasia. Animals were hum anely euthanized by decapitation 2-5 hoursMale Pubertal Assay (2012) / Page 7 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None 

### post-dose, in the same order as they were dosed. After euthanasia and prior to tissue collection, trunk blood was collected, and seru m was obtained. All serum samples were stored at or below

− 70°C. Serum samples were shippe d on dry ice to AniLytics Inc. (Gaithersburg, MD) for analysis.

a. Hormone Analysis: Testosterone, T 4 

### , and TSH were measured in serum using radioimmunoassays (RIAs). Samples were as sayed in duplicate and in conjunction with multiple quality control samples.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER
X Calcium X Albumin
X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus Total cholesterol
X Potassium Globulins X Sodium Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Albumin /globulin ratio (calculated) Lactic acid dehydrogenase (LDH) Bile acids X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) X Sorbitol dehydrogenase (SDH) X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in male rats based on guideline 890.1500.


### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed and grossly observed fr om all study animals. The (XX) organs, in addition, were subjected to histological examination.

UROGENITAL OTHER XX Testes (left and right separately)*+ XX Thyroid a * + XX Epididymides (left and right separately)*+ X Liver* X Seminal vesicle plus coagulating glands (w ith and without fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prostate* X Levator ani/bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* + a Thyroid was weighed post-fixation. * Weights required base d on guideline 890.1500. + Histopathological examination re quired based on guideline 890.1500.


### Left kidney and thyroid (with a ttached trachea) were fixed in 10% neutral buffered formalin for at least 24-hours. Following fixation, the th yroid was dissected free of the trachea andMale Pubertal Assay (2012) / Page 8 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None 

### weighed. Left testis and left epididymis we re each placed in Bouin’s fixative for 18- to 24- hours and then washed in 70% histology grad e alcohol. After fixation, tissues were transferred to 70% histology grade alcohol, routinely processed into paraffin blocks, sectioned, and stained with hematoxylin and eosin. Histologically -processed tissues (thyroid, left kidney, left epidi dymis, and left testis) from all animals were microscopically evaluated. Two sections of each of the 2 lobe s of the thyroid were examined and evaluated for follicular cell height and colloid area. A 5- point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used, and the thyroids were evaluated relative to photomicrographs supplied by the U.S. EPA.

II. RESULTS

A. MORTALITY: All animals survived to scheduled sacrifice.

B. CLINICAL SIGNS OF TOXICITY: No treatment-related clin ical signs were observed. Rough coat was observed on at least 1 day in 3 control animals, 4 animals in the 62.5 mg/kg/day group, and 3 animals in the 125 mg/k g/day group. However, this finding is considered incidental as these animals appeared normal on all other days of the study. In addition, 1 animal control animal exhibited scab s on the throat and near the left eye on at least 1 day. All other rats a ppeared normal throughout the study.

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight gains, age of attainment of PPS, weight at PPS, and proportion/incidence of unseparated preputia are presented in Table 2 below. All values in the treated groups were similar to controls.

The following Guideline performance criteria we re not met: (i) CV=6.1% for age at PPS (top of acceptable range: 5.67%); (ii) CV=13.3% for weight at PPS (top of acceptable range: 7.57%); (iii) CV=8.1% for final body weight (t op of acceptable range: 7.47%); and (iv) mean weaning weight was 64.1 g while the acceptable range was 45.472-59.812 g.Male Pubertal Assay (2012) / Page 9 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control 62.5 mg/kg/da y EPTC 125 mg/kg/day EPTC

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Age at PPS (PND) U 15 44.9 2.7 6.1 16 44.4 1.7 3.8 15 45.1 2.1 4.7
A 15 44.9 --- --- 16 44.4 --- --- 15 45.1 --- --- Body weight at PPS (g) U 15 239.6 31.9 13.3 16 226.2 25.3 11.2 15 232.9 29.4 12.6
A 15 239.3 --- --- 16 226.3 --- --- 15 233.2 --- ---

Age at partial PPS (PND) U 15 40.9 1.3 3.3 16 40.8 1.2 3.0 15 41.3 1.1 2.7

A 15 40.9 --- --- 16 40.8 --- --- 15 41.3 --- ---

Initial body weight (PND 23; g) U 16 64.1 4.3 6.6 16 62.8 7.1 11.4 16 64.7 4.8 7.5

A 16 64.1 --- --- 16 62.8 --- --- 16 64.7 --- ---
Final body weight (g) U 16 311.3 25.1 8.1 16 299.6 28.6 9.5 16 297.1 34.8 11.7
A 16 311.3 --- --- 16 299.6 --- --- 16 297.1 --- ---

Final body weight (% of control) U 16 --- --- --- 16 96.2 --- --- 16 95.4 --- ---

A 16 --- --- --- 16 96.2 --- --- 16 95.4 --- ---

Body weight gain (final – initial; g) U 16 247.1 27.0 10.9 16 236.8 26.5 11.2 16 232.4 32.0 13.8

A 16 247.1 --- --- 16 236.8 --- --- 16 232.4 --- --- Proportion unseparated (#/N) 1/16 0/16 1/16
a Data were obtained from Table 16 on page 40 of the st udy report. No statistical differences were detected. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 N Number of animals examined SD Standard Deviation CV Coefficient of Variation



### D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. The LABC weights showed a linear trend of decreased (p ≤ 0.05) weight with incr easing dose; however, no statistically significant effect was detected between the treated gr oups and control using Dunnett’s test. No other statis tically significant effect or tr end was detected on the weight of the sex tissues or any other organs examined.

The unadjusted values for all organ weights in the control group were within the acceptable range of the performance criteria provided in the Guideline (8 90.1500), with the exception of: (i) mean kidney weight of 2.06 g (accep table range: 2.242-3.050 g); (ii) mean LABC weight of 0.421 g (acceptable range: 0.447-0.855 g) ; (iii) CV=22.9% for seminal vesicles with coagulating gland and fluid (top accep table range: 21.06%); and (iv) CV=22.1% for testis (top acceptable range: 17.62%).Male Pubertal Assay (2012) / Page 10 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control 62.5 mg/kg/da y EPTC 125 mg/kg/day EPTC

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 16 14.45 1.21 8.4 16 14.15 2.00 14.2 16 14.01 1.86 13.3
A 16 14.45 - - 16 14.15 - - 16 14.01 - -
R 16 4.58 0.20 4.3 16 4.65 0.32 6.8 16 4.63 0.27 5.7

Kidneys (g) U 16 2.06 0.19 9.2 16 1.99 0.21 10.6 16 2.03 0.21 10.4
A 16 2.06 - - 16 1.99 - - 16 2.03 - -
R 16 0.66 0.04 5.7 16 0.66 0.03 5.0 16 0.67 0.03 3.8

Pituitary (mg) U 16 8.8 1.2 13.9 16 8.8 1.8 20.9 16 8.7 1.6 18.4
A 16 8.8 - - 16 8.8 - - 16 8.7 - -
R 16 2.8 0.4 15.4 16 2.9 0.6 19.1 16 2.9 0.3 10.1

Adrenals (mg) U 16 43.7 4.4 10 16 45.4 6.1 13.5 16 43.3 5.6 12.9
A 16 43.7 - - 16 45.4 - - 16 43.3 - -
R 16 14 1.8 13.2 16 15.0 1.6 10.8 16 14.4 1.5 10.7

Seminal vesicle + coagulating gland, with fluid (mg) U 16 548.3 125.5 22.9 16 557.9 93.2 16.7 16 515.7 125.6 24.4

A 16 548.3 - - 16 557.9 - - 16 515.7 - -

Seminal vesicle + coagulating gland, without fluid (mg) U 16 318.5 64.0 20.1 16 321.3 46.0 14.3 16 315.5 76.8 24.3

A 16 318.5 - - 16 321.3 - - 16 315.5 - -

Ventral prostate (mg) U 16 268.2 47.3 17.6 16 260.0 47.6 18.3 16 247.1 49.7 20.1
A 16 268.2 - - 16 259.9 - - 16 247.1 - - Dorsolateral prostate (mg) U 16 147.5 29.3 19.9 16 144.5 30.8 21.3 16 137 24.8 18.1
A 16 147.5 - - 16 144.5 - - 16 137 - -
LABC (mg) b U 16 420.8 58.2 13.8 16 412.6 63.0 15.3 16 382.7 40.9 10.7
A 16 420.9 - - 16 412.6 - - 16 382.6 - - Epididymis, left (mg) U 16 202.3 18.4 9.1 16 200.5 22.9 11.4 16 203.9 13.7 6.7
A 16 202.3 - - 16 200.5 - - 16 203.9 - - Epididymis, right (mg) U 16 204.8 21.8 10.7 16 206.0 26.3 12.8 16 209.2 23.1 11.0
A 16 204.8 - - 16 206.0 - - 16 209.2 - - Testis, left (mg) U 16 1530 153.7 10.0 16 1480.6 161.6 10.9 16 1495.6 132.2 8.8
A 16 1530.1 - - 16 1480.7 - - 16 1495.4 - - Testis, right (mg) U 16 1726.6 584.2 33.8 16 1494.9 150.5 10.1 16 1561.1 268.4 17.2
A 16 1726.7 - - 16 1495.1 - - 16 1560.9 - - Thyroid, fixed (mg) U 16 17.7 3.3 18.8 16 17.6 4.0 22.9 16 17.1 2.0 12.0
A 16 17.7 - - 16 17.6 - - 16 17.1 - - a Data were obtained from Table 17 on pages 42-43 of the study report. No differences (p ≤ 0.05) were noted between treated groups and controls. b A significant (p ≤ 0.05) linear trend was noted. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 R Organ-to-body weight ratio (relative to body weight) SD Standard Deviation CV Coefficient of Variation



### E. CLINICAL CHEMISTRY AND HORMONE LEVELS: Clinical chemistry and hormone levels are presented in Table 4. Serum T 4 

### levels were increased (p<0.05) by 12%Male Pubertal Assay (2012) / Page 11 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None 

### in the 125 mg/kg/day group, but the concentra tion was within the reported normal range. Serum TSH and testosterone leve ls in the treated groups were similar to controls.

The following clinical chemistry parameters differed (p<0.05) in the treated groups compared to controls: (i) chloride increased by 2% at both doses; (ii) SDH increased by 28 and 40% at 62.5 and 125 mg/kg/da y respectively; and (iii) al bumin decreased by 5% at 125 mg/kg/day. These differences were minor and were within the reported normal range.

The results for serum T 4 

### , TSH, and testosterone in th e control group were within the acceptable range of the performance criteria provided in the Guideline (890.1500), with the exception of: mean serum TSH level of 3.92 ng/mL (acceptable range: 4.212–24.112 ng/mL); and CV=72.1% for serum TSH (top of acceptable range: 58.29%).

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control 62.5 mg/kg/da y EPTC 125 mg/kg/day EPTC

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Hormones
Serum T 4 , Total (μg/dL) 16 4.81 0.72 15.0 16 4.49 0.62 13.9 16 4.22* ( ↓ 12) 0.84 19.8

Serum TSH (ng/mL) 16 3.92 2.82 72.1 16 4.18 2. 65 63.4 16 2.78 1.26 45.2

Serum
testosterone (ng/mL) 16 2.45 1.59 65.0 16 2.89 2. 44 84.6 16 1.54 1.43 93.2Male Pubertal Assay (2012) / Page 12 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None

TABLE 4. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control 62.5 mg/kg/da y EPTC 125 mg/kg/day EPTC

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Clinical Chemistry Sodium (mEq/L) 16 143 6 4.3 16 144 2 1.5 16 144 2 1.0

Potassium (mEq/L) 16 8.1 0.5 6.4 16 8.3 0.6 7 16 8 0.6 8.1

Chloride (mEq/L) 16 105 1 1.2 16 107*** ( ↑ 2) 2 1.5 16 107*** ( ↑ 2) 1 1.4

Calcium (mg/dL) 16 10.3 0.3 3.2 16 10.3 0.5 5 16 10.3 0.2 2.4
Phosphorus (mg/dL) 16 9.7 0.9 9.6 16 9.9 0.9 9 16 9.4 0.8 8.3
AST (U/L) 16 228 40 17.4 16 263 72 27.4 16 235 39 16.6 ALT (U/L) 16 102 12 11.8 16 108 18 16.5 16 105 10 9.3 GGT (U/L) 16 0.1 0.3 273 16 0.4 0.8 186 16 0.3 0.9 279 ALP (U/L) 16 523 98 18.8 16 521 95 18.2 16 475 96 20.1
BUN (mg/dL) 16 11 2 16.4 16 11 2 16.8 16 9 2 21.2

Creatinine (mg/dL) 16 0.3 0.1 18.3 16 0.4 0.1 19.1 16 0.4 0 13

Total Bilirubin (mg/dL) 16 0.02 0.04 215 16 0.04 0.06 165 16 0.04 0.06 165

SDH (U/L) 16 25 4 14.2 16 32** ( ↑ 28) 9 26.2 16 35*** ( ↑ 40) 5 15.0

Total Protein (g/dL) 16 5.8 0.2 3.5 16 6 0.2 4.1 16 5.7 0.2 4.1

Albumin (g/dL) 16 4.2 0.2 5.3 16 4.3 0.2 4.2 16 4.0* ( ↓ 5) 0.2 5.0
a Data were obtained from Table 18-19 on pages 45-46 of the stud y report. Percent difference from controls (calculated by reviewers) is presented parenthetically. SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05. * Significantly different fro m controls at p<0.01. * Significantly different fro m controls at p<0.001.



### F. GROSS PATHOLOGY: There were no macroscopic findings reported in any animal.

G. HISTOPATHOLOGY: The incidences of histopathologi cal findings of the thyroid gland are presented below in Table 5; follicular ce ll shape was not reported. The colloid area and follicular cell height of the thyroid in the treat ed groups were similar to the control group.Male Pubertal Assay (2012) / Page 13 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None

TABLE 5. Incidence of Histopathol ogical Lesions of the Thyroid Gland a

Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height (Increase)

Grade b Incidence Grade b Incidence O E O E

Vehicle Control 1 0 16 1 0 16 2 2 16 2 1 16 3 13 16 3 13 16 4 1 16 4 2 16 5 0 16 5 0 16

62.5 mg/kg/day EPTC 1 0 16 1 0 16 2 0 16 2 1 16 3 15 16 3 15 16 4 1 16 4 0 16 5 0 16 5 0 16

125 mg/kg/day EPTC 1 0 16 1 0 16 2 0 16 2 3 16 3 13 16 3 13 16 4 3 16 4 0 16 5 0 16 5 0 16 a Data were obtained from Table 21 on page 48 of the study report. b A 5-point grading scale (1 = shortest/s mallest; 5 = tallest/largest) was used. O No. Observed E No. Examined


### The incidences of histopathological findings of the testes, epididymides, and kidneys are presented in Table 6, and there were no treatment-related findings.

TABLE 6. Incidence of Histopathological Lesi ons of the Testis, Epididymides, and Kidney a

Findings Dose Level Vehicle Control 62.5 mg/kg/day EPTC 125 mg/kg/day EPTC

# Observed # Examined # Observed # Examined # Observed # Examined

Testis
Seminiferous tubules, mild dilatation 0 16 0 16 1 16
Epididymis

Minimal aberrant cells in tubules lumen 0 16 8 16 3 16

Minimal interstitium inflammation 9 16 11 16 9 16
Kidneys
Minimal nephropathy 7 16 9 16 11 16
Cysts present 1 16 2 16 0 16 a Data were obtained from Appendix X on pages 141-147of the study report.Male Pubertal Assay (2012) / Page 14 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Administration of EPTC to intact juvenile/ peripubertal male rats at a dose that met, but did not exceed the maximum tolerated dose, did not cause changes in any endpoints eval uated to suggest a disruption in pubertal development or thyroid function.

B. AGENCY COMMENTS: No treatment-related related e ffects were observed on survival, clinical signs, body weights, fi nal body weight, body weight gain s, organ weights, clinical chemistry, gross patholo gy, or renal histology.

The following clinical chemistry parameters differed (p<0.05) in the treated groups compared to controls: (i) chloride increased by 2% at both doses; (ii) SDH increased by 28 and 40% at 62.5 and 125 mg/kg/da y respectively; and (iii) al bumin decreased by 5% at 125 mg/kg/day. These differences were minor, within the reported normal range, and were not considered adverse.

No treatment-related related effects were obs erved on body weight or age at PPS, age at partial PPS, proportion/incidence of unseparated preputia, an drogen-dependent tissue organ weights (except LABC), or serum testosterone and thyroid hormone levels. There was a small (12%) decrease in serum T4 at 125 mg/kg bw. However, the decrease was still within the normal range reported and there were no effects on the other thyroid hormones. The colloid area and follicular cell height of the t hyroid in the treated groups were similar to the control group. The incidences of histopathological findings of the testes and epididymides in the treated groups were similar to controls.

The LABC weights showed a linear trend of decreased (p ≤ 0.05) weight with increasing dose; however, no statistically si gnificant effect was detected between the treated groups and the control group. No other statistically sign ificant effect or trend was detected for the sex tissues or any other organ weighed. Th erefore, this linear trend was considered incidental.

Although no toxicity was noted at the high dos e (125 mg/kg/day) and terminal body weight was only decreased by 5% (NS), re lative to the control, the high dose is considered adequate for purposes of this assay based on data from a range-finding study using juvenile males in which dosing at 200 and 400 mg/kg/day resulted in body weight decreases of 14 and 15%, respectively.

In this study, there was no evidence to suppor t an effect of EPTC on pubertal development and thyroid function at do ses up to 125 mg/kg/day.

C. STUDY DEFICIENCIES: The following deficiency was no ted that is not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The stability data from Smithers Vi scient, LLC. Study No. 12791-6162 were not submitted for independent evaluation.Male Pubertal Assay (2012) / Page 15 of 15 EPTC / 041401 OCSPP 890.1500/ OECD None 

### Additionally, a number of Guideline performance criteria were not met:

 CV=6.1% for age at PPS (top of acceptable range: 5.67%)

 CV=13.3% for weight at PPS (top of acceptable range: 7.57%)

 CV=8.1% for final body weight (top of acceptable range: 7.47%)

 Mean weaning weight was 64.1 g while the acceptable range was 45.472-59.812 g

 Mean kidney weight of 2.06 g (acceptable range: 2.242-3.050 g)

 Mean LABC weight of 0.421 g ( acceptable range: 0.447-0.855 g)

 CV=22.9% for seminal vesicles with coagula ting gland and fluid (top acceptable range: 21.06%)

 CV=22.1% for testis (top acceptable range: 17.62%)

 Mean serum TSH level of 3.92 ng/mL (acceptable range: 4.212–24.112 ng/mL)

 CV=72.1% for serum TSH (top of acceptable range: 58.29%)

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-13-2012 (MRID 48616401)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 1/6/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 1/17/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 1/25/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 1/25/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).EPTC I 041401 

### Primary Reviewer: Gregory Akerman 

### Health Effects Division Secondary Reviewer: John Liccione, Ph.D

. 

### Health Effects Division

DAT A EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550



### PC CODE: 041401 

### DP BARCODE: D397177



### TXR#: 0051762 

### CAS No.: 759-94-4



### TEST MATERIAL (PURITY): EPTC ( > 99.9% a.i.)



### SYNONYMS:

CITATION:

SPONSOR: S-ethyl N
, N-dipropylcarbamothioate

Wagner
, H

. (2011). EPTC: H295R Steroidogenesis Assay. CeeTox, Kalamazoo
, MI. Study No. 9 l 42V-l 00349STER, November 10
, 2011. MRID 48616401. Unpublished.

Gowan Company, 370 S. Main Street, Yuma, AZ



### TEST ORDER#: E DSP-041401-64



### EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48616401)
, H295R cells cultured 

### in vitro in 24-well plates were incubated with EPTC ( > 99.9% purity
, Batch # Pl 105096) at concentrations of 100
, 10
, 1
, 0.1
, 0

. 01
, 0.001
, and 0.0001 μM for 48 hours in triplicate for four independent experiments

. Run 1 was not analyzed because one of the forskolin concentrations on the quality control plate was incorrect (3 μM was used instead of 1 μM). Dimethyl s ulfoxide (DMSO) was used as a vehicle
, at a final concentration of 0

. 05%.

Testosterone and estradiol levels were measured using HPLC-MS / MS. A Quality Control (QC) plate was run concurrently w ith each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels

. The positi ve controls included a known inhibitor (prochloraz) and inducer (forskolin) of estradiol and
testosterone production.

All gu id eline acceptabilit y recommendations and requirements were met , including lack of cytotoxicity, adequate production of testosterone and estradiol
, acceptable reproducibility (low coefficients of variation, %C V)
, and appropriate induction and inhibition with positive controls

.

Testosterone concentration was not affected b y EPTC. In three independent runs , statistically significant inhibition or induction of testosterone production was not observed. Decreases ( p :S 0.05) of9

- 10 % in es tr adio l were observed at EPTC concentrations of0.01
, 0

. 001
, and 0.0001 μMin Run 3. When the estradiol results in Run 3 were normalized to percent viability, the inhibition of estradiol at 0.0001
, 0

. 001
, and 0.01 μM was not statistically significant. InSteroidogenesis Assay (2011) / Page 2 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None 

### addition, these decreases were minor and not re produced in the other two runs. However, increases (p ≤ 0.05) of 10–15% in estradiol concentrations were obser ved at the 100 μM dose of EPTC in Run 3 (with and without normalization to viability) and in Run 4. Although the effect was slight, it was statistically si gnificant in two of three runs a nd was reproducible. Therefore, it is concluded that EPTC at 100 μM may induce of estradiol production.

Based on the hormone responses in each of the th ree independent runs, EPTC treatment resulted in statistically significant and re producible increases in estradiol production, but did not result in statistically significant and reproducible alterations in testosterone production.

The assay satisfies the EDSP Tier 1 Test Order require ments for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated GLP Compliance, Quality Assurance, and Data Confidentiality statements were provided.Steroidogenesis Assay (2011) / Page 3 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox Location: Kalamazoo, MI Study Director: Hilary Wagner, M.S. Other Personnel: Colleen Toole, Ph.D., Director of Project Management Karen Rutherford, B.S., Direct or of Laboratory Operations David Blakeman, B.S., Senior Sc ientist/Endocrine Group Leader Briana Wallace, B.S., Lead Cell Culture Scientist Steven McColley, M.S., Scientist Cameron Haines, B.S., Scientist Francisca Wong, M.S., Scientist Joey Burgam, B.S., Associate Scientist Lisa Blakeman, B.S., Associate Scientist Study Period: July 15, 2011 through November 10, 2011



### 2. Test Substance: EPTC Description: Liquid Batch # (expiration date): P1105096 (February 4, 2015) Purity: >99.9% Solubility: Soluble at tested concentrations in DMSO Vapor pressure: 4500 mPa at 25°C Stability: Not reported Storage conditions: Room temperature CAS #: 759-94-4 Molecular weight: 189.3 Structure:

N S O CH

3

CH 3 CH 3



### 3. Positive Control: Forskolin Description (molecular weight): White powder (410.50) Source: Sigma Aldrich (St. Louis, MO) Lot # (expiration date): 109K50571V (July 2016) Purity: 98% Solubility (in solvent): Soluble at tested concentrations in DMSO Storage conditions: Room temperature CAS #: 66575-29-9


### 4. Negative Control: Prochloraz Description (molecular weight): White powder (376.67) Source: Sigma Aldrich (St. Louis, MO) Lot # (expiration date): SZE6220X (August 8, 2012) Purity: 99.1% Solubility (in solvent): Soluble at tested concentrations in DMSO Storage conditions: Room temperature CAS #: 67747-09-5Steroidogenesis Assay (2011) / Page 4 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None



### 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) Description (molecular weight): Clear liquid (78.13) Source: Sigma-Aldrich (St. Louis, MO) (catalog # not provided) Lot # (expiration date): RNBB7617 (Not provided) Purity: Not provided Storage conditions: Not provided CAS #: 67-68-5 Justification for choice of solvent: Not provided

Final concentration in assay: 0.05% v/v



### 6. Other Materials: 22R-Hydroxycholesterol Description (molecular weight): White powder (402.65) Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 089K4132, 060M4098 (not provided) Purity: 99.0% Storage conditions: Room temperature Solvent used: Ethanol, final concentratio n in assay, 0.025% v/v CAS #: 17954-98-2



### 7. Stock Medium: Dulbecco’s modified Eagle’s medium/nutrient mixture F12 Ham Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 021M8304 (not provided)
HEPES: 15 mM; Sigma-Aldrich (St. Louis, MO); Lot # 021M8304 Sodium bicarbonate: Sigma-Aldrich (St. Louis, MO); Lot # 068K0105; purity not reported Nu-Serum: 2.5%; Becton-Dickinson (Franklin Lake s, NJ); Catalog # 355500; Lot # 81515; tested for background hormone concentr ations by performing laboratory ITS+premix: Becton-Dickinson (Franklin Lakes, NJ); Catalog # 354352; Lot # 05245, 09233



### 8. Test Cells: H295R human adrenocortical carcin oma cells (ATCC CLR-2128; Lot # 7635054) at passage 7.5 – 8.5 were incubated in the stock medium, supplemented with 10 μM 22R-hydroxycholesterol in ethanol at th e time of plating, dosing, and harvest. Incubation conditions were at 5% CO 2 

### and 37 ± 2°C for approximately 24 hours prior to exposure.
The following performance criteria were met (indicated by an “x”): X Cell passage identifier. Cell Passage #: 7.5-8.5 X Cells frozen down at passage 5 X Frozen cells cultured for 4 additional passages X Total number of passages does not exceed 10



### B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: A hormone assay interference test was not performed.

b. Hormone Extraction: See Section on “Hormone Measurement System.”

c. Laboratory Proficiency Test: No laboratory proficiency te st data were provided.

2. Test Solutions: EPTC was dissolved in DMSO to make a 200 mM stock and then serially diluted in DMSO. 22R-hydroxycholesterol was dissolved in ethanol to make a 40 mMSteroidogenesis Assay (2011) / Page 5 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None 

### stock and then diluted in suppl emented medium to a final concentration of 10 μM. EPTC was then diluted in supplemented medium containing 10 μM 22R- hydroxy-cholesterol to prepare mastermix solutions. Forskolin and pr ochloraz were dissolved in DMSO to make 100 mM solutions and then serially diluted in DMSO. Forskolin and prochloraz were then diluted in supplemented medium containing 10 μM 22R-hydroxy-cholesterol. When added to the cell culture plates, these dilutions yielde d final concentrations of 1 μM and 10 μM for forskolin, 0.1 μM and 1 μM for proc hloraz, and 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μM for EPTC. The final concentra tion of DMSO in the medium was held constant at 0.05% (v/v). The final ethanol concentration in th e supplemented medium was 0.025% (v/v). No precipitation was visible.

3. Cell Plating and Preincubation: Cells were maintained in the Stock Medium described above. It was reported that hormone con centrations in undiluted Nu-Serum were 3754 pg/mL testosterone and 3846 pg/mL estradio l. H295R cells were grown for five passages, frozen in liquid nitrogen, then thaw ed and cultured for seven or eight additional passages prior to use in the assay. The culture medium was supplemented with 10 uM 22R-hydroxycholesterol at the time of plating, do sing, and harvest. The concentration of 22R-hydroxycholesterol was chosen based on labora tory proficiency experiments previously conducted at the performing laboratory. The ce lls were plated into wells of a 24-well cell culture plate at a density of approximately 300,000 cells/mL. The cells were then placed into a 5% CO 2 

### incubator at 37 ± 2°C for approximately 24 hours prior to chemical exposure. Prior to dosing, the cells were checked microscopically for attachment and proper morphology.

4. Exposure: The medium was removed from the ce lls and replaced with 1 mL of medium containing 10 μ M 22R-hydroxycholesterol and concentra tions of EPTC in triplicate, according to the schematic presented in Table 1.

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to EPTC (Final Co ncentrations in μM). a 1 2 3 4 5 6 A DMSO DMSO DMSO 0.1 0.1 0.1 B 100 100 100 0.01 0.01 0.01 C 10 10 10 0.001 0.001 0.001 D 1 1 1 0.0001 0.0001 0.0001 a Data were obtained from page 13 of the study report.



### A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiol production). The QC plate was prepared and dosed in the same manner with either forskolin or prochloraz, according to the schematic presented in Table 2.

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM). a 1 2 3 4 5 6 A Blank b Blank Blank Background c Background Background B DMSO DMSO DMSO DMSO + MeOH d DMSO + MeOH DMSO + MeOH C Forskolin 1 μM Forskolin 1 μM Forskolin 1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM Prochloraz 0.1 μMSteroidogenesis Assay (2011) / Page 6 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None

D Forskolin 10 μM Forskolin 10 μM Forskolin 10 μM Prochloraz 1 μM Prochloraz 1 μM Prochloraz 1 μM a Data were obtained from page 13 of the study report. b Blank wells received medium contai ning 10 μM 22R-hydroxycholesterol. c Background wells received medium without 22R-hydroxycholesterol). d MeOH = methanol was added to these wells for 30 mi nutes at room temperature following medium removal.



### Following dosing, the plates were incubated for 48 ± 2 hours under the conditions previously described. After the 48 ± 2 hour exposure, each well was examined under the microscope. Images were taken of the vehi cle control wells, as we ll as the two highest concentrations. Cytotoxicity was not observed. The media was collected from all wells in two equal aliquots and stored at -80 ± 10°C until shipment to the analytical laboratory for hormone measurement.

5. Cell Viability/Cytotoxicity Assay: After media removal, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte trazolium bromide (MTT) assay. On the QC plates, wells designated to receive methanol (control wells for cell death measurements) were rinsed twice with phos phate buffered saline (PBS; Gibco; Cat. # 10010; Lot # 979431), and then incubated in methanol (Sigma; Ca t. # 494437; Lot # SHBB0973V) for 30 minutes at room temperature. After the methanol incu bation, the methanol-treated wells were rinsed again with PBS three times. Following medi a removal and/or methanol treatment, 0.5 mL of 0.5 mg/mL MTT (Sigma; Cat. # M5655; Lo t # MKBG1611V) solution in supplemented medium containing 10 μM 22R-hydroxycholesterol was added to each well of the test chemical and QC plates. The plates were incubated at 37±2°C in a 5% CO 2 

### incubator for 3 hours. Following the 3 hour incubation, th e MTT solution was removed, and 0.5 mL of isopropanol was added to each well. Plates were incubated at room temperature for 20 minutes with shaking. Following this incubation, absorbance at 570 nm and 650 nm were measured on a Packard Fusion plate reader . The absorbance at 650 nm was subtracted from the absorbance at 570 nm to calculate the MTT value for each well. The change in cell viability was determined by comparing treated we lls to the solvent control (SC) wells. A greater than 20% reduction in cell viability was considered evidence of cytotoxicity.

6. Hormone Measurement System: Testosterone and estradio l levels were measured at OpAns, LLC (Durham, NC). Testosterone and estradiol were extracted from H295R supplemented medium by liquid-liquid extract ion using methyl tert -butyl ether (MTBE) after the addition of [ 2 

### H 5 

### ]-testosterone and [ 2 

### H 5 

### ]-estradiol as internal standards. Extracts were analyzed by HPLC-MS/MS using positive ion multiple reaction monitoring. This method was validated over the range 100 to 100,000 pg/mL for testosterone and 10 to 10,000 pg/mL for estradiol (OpAns Document # OPR-OPP-0006). The lower limit of quantification (LLQ) was 100 pg/mL for testos terone and 10 pg/mL fo r estradiol using a 300 μL aliquot of H295R supplemented medium . Freshly prepared calibration standards (n = 7) were prepared in supplemented me dium for each run. QC samples spiked in supplemented medium at three concentrations (2 replicates per concentration), were included with each run. No study sample s were re-analyzed during this study.

The following performance criteria were met (indicated by an “x”): X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

X Spiked sample recovery acceptable fo r two concentrations of testosterone and estradio l (mean measured amount from triplicate samples within 30% of nominal concentration)Steroidogenesis Assay (2011) / Page 7 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None

NA Hormone cross-reactivity (antibody-based assays only; ≤ 30% of basal production of the respective hormone) X Solvent control within 75% range be low maximum response on standard curve X Test compound tested for interfer ence with meas urement system



### C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations for testosterone and estradiol were calculated for each concentrati on of EPTC, reference chemicals, the solvent controls, blanks, and background wells. Rela tive changes in testosterone and estradiol production were calculated us ing the equation below:

Relative Change = [Hormone] in each well ÷ [Hormone] of mean solvent control

For forskolin induction of testosterone, relative change was calculated both with and without subtracting the background horm one production from the forsko lin-treated wells, and blank and SC wells. Background hormone production wa s calculated from thre e wells with cells on the QC plate that recei ved no 22R-hydroxycholesterol at the time of exposure.

Normality of the data was evaluated using Shapiro-Wilk's test. Homogeneity of the variances between the tr eatment groups was evaluated using Le vene's test. If the p-values were greater than 0.05 in both tests, statisti cal significance between each treatment group and the control group was evaluated using Dunnett's test. If the p-values were less than or equal to 0.05 in either the normality or th e homogeneity test, a log transformation was performed on the data to attempt to approxi mate a normal distribution. If (following the transformation) p-values were greater than 0.05 in the normality and homogeneity tests, Dunnett's test was performed on the transformed data to evaluate st atistical significance between each treatment group and the control group. If follo wing the log transformation, pvalues were less than or equal to 0.05 in ei ther the normality or the homogeneity test, the non-transformed data set was analyzed us ing the nonparametric Kruskal-Wallis test followed by Dunn's test to evaluate statistic al significance between each treatment group and the control group. Data analysis at th e analytical laborato ry (OpAns, LLC) was conducted in Microsoft Excel. No additional so ftware for statistical analyses or graphing was specified. The reviewers cons ider these analyses acceptable.

II. RESULTS

A. TEST COMPOUND: Pr ecipitation of the test com pound was not observed at EPTC concentrations up to 100 μ M. The %CVs for absolute hormone concentrations for the SC replicates within the test pl ates were 3.1–6.0% for testostero ne and 0.9–3.0% for estradiol. The between plate CV for SC based on absolute concentrations (calcu lated by reviewer) was 19.5% for testosterone and 11.3% for estrad iol. These data met the recommended performance criteria of ≤ 30%.

The Sponsor stated: “Four independent runs of the steroidogenesis assay were conducted. Run 1 was not analyzed because one of the fo rskolin concentrations on the QC plate was incorrect (3.33 μM was used instead of 1 μM). Data from Run 1 will not be presented in this report as the media was not analyzed for testosterone and estradiol.” The concentrations of estradiol a nd testosterone observed in th e other three assay runs are summarized in Table 3 and graphica lly depicted in Figures 1 and 2.Steroidogenesis Assay (2011) / Page 8 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None



### Testosterone concentration was not affected by EPTC. In three independent runs of the assay, statistically significant inhibition or induction of te stosterone production was not observed. Decreases (p ≤ 0.05) of 9–10% in estradiol we re observed at the 0.01, 0.001, and 0.0001 μM doses in Run 3. However, when the estradiol results in Run 3 were normalized to percent viability, the inhibition of estradiol at 0.0001, 0.001, and 0.01 μM was not statistically significant. Increases (p ≤ 0.05) of 10–15% in estradiol was observed at the 100 μM dose in Run 3 (with and without normalizat ion to viability) and in Run 4. No effect was observed on testosterone or estradiol at other EPTC concentrations in Run 2.

TABLE 3. Mean (±SD) Hormone Concen trations Following Treatment with EPTC for 48 Hours. a Nominal Concentration (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b Statistical Significance
Testosterone (pg/mL) Fold Difference DMSO 2743 1845 2441 NA NA NA NA NA NA 0.0001 3031 1810 2447 1.11 0.98 1.00 1.03 0.07 — 0.001 2694 1787 2421 0.98 0.97 0.99 0.98 0.01 — 0.01 2820 1772 2339 1.03 0.96 0.96 0.98 0.04 — 0.1 2903 1846 2420 1.06 1.00 0.99 1.02 0.04 — 1 2818 1855 2433 1.03 1.01 1.00 1.01 0.02 — 10 2845 1805 2387 1.04 0.98 0.98 1.00 0.03 — 100 2850 1882 2473 1.04 1.02 1.01 1.02 0.02 — Estradiol (pg/mL) Fold Difference DMSO 233 197 246 NA NA NA NA NA — 0.0001 222 179 231 0.95 0.91* 0.94 0.93 0.02 Run 3 0.001 213 178 231 0.91 0.90* 0.94 0.92 0.02 Run 3 0.01 229 179 237 0.98 0.91* 0.96 0.95 0.04 Run 3 0.1 230 187 231 0.99 0.95 0.94 0.96 0.03 — 1 232 184 250 1.00 0.94 1.02 0.99 0.04 — 10 238 196 251 1.02 1.00 1.02 1.01 0.01 — 100 276 217 282 1.19 1.10* 1.15* 1.15 0.05 Runs 3 and 4 a Data were obtained from pages 25-26 and 33-35 of the stud y report. The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol. b Calculated by the reviewers. NA Not applicableSteroidogenesis Assay (2011) / Page 9 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None



### FIGURE 1. Change in Testosterone Production Relative to EPTC Concentration.Steroidogenesis Assay (2011) / Page 10 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None 

### FIGURE 2. Change in Estradiol Produc tion Relative to EPTC Concentration.Steroidogenesis Assay (2011) / Page 11 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None


### B. CYTOTOXICITY: Less than 5% cytotoxicity wa s noted in the reference and EPTC-treated wells (96–107% cy totoxicity, unrelatedto dose), except in the methanoltreated wells. Data are summarized in Tabl e 4 and depicted graphi cally in Figure 3.

TABLE 4. Mean (±SD) MTT Cell Viability (% SC) Resu lts after Treatment with Forskolin, Prochloraz, or EPTC for 48 Hours. a Compound Concen. (μM) Cell Viability – Run 2 Cell Viabilit y – Run 3 Cell Viability – Run 4 Mean SD Mean SD Mean SD Blank NA 99.4 10.54 100.1 4.93 103.0 1.84 Background NA 102.5 5.95 98.7 0.69 104.0 1.76 SC + Methanol NA 4.9 0. 99 5.7 0.64 8.3 1.79 Forskolin 1 103.9 3.88 97.2 2.20 107.3 0.91 Forskolin 10 105.1 4.90 97.5 2.67 106.7 4.11 Prochloraz 0.1 100.7 1.89 97.8 1.79 101.7 2.17 Prochloraz 1 101.1 2.01 96.1 2.79 105.0 4.29 EPTC 0.0001 99.8 1.21 97.6 2.22 100.7 1.32 EPTC 0.001 98.6 1.07 98.7 2.04 100.1 2.12 EPTC 0.01 96.6 0.65 96.3 0.96 102.3 2.45 EPTC 0.1 99.6 1.85 100.8 1.81 101.2 0.66 EPTC 1 97.6 1.00 97.5 1.66 98.5 1.53 EPTC 10 99.4 1.07 99.1 1.36 100.2 0.38 EPTC 100 98.2 0.61 98.4 3.45 100.2 3.25 a Data were obtained from pages 19-20 of the study report. NA Not applicableSteroidogenesis Assay (2011) / Page 12 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None 

### FIGURE 3. MTT cell viability results for EPTC-treated cells.Steroidogenesis Assay (2011) / Page 13 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None 

### C. QC PLATE: The hormone concentrations after expos ure to the reference chemical, SC, blank, and background samples, as well as the fold-difference change relative to SC (individual and mean ± SD) for the three suitable assay runs, are presen ted in Table 5. The minimum basal hormone production levels ( 500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both blank and SC wells . Basal hormone production in SC wells was >17-fold minimum detection level (MDL) for te stosterone and >17-fold MDL for estrogen. Compared to SC, 10 μM forskolin on average induced testosterone by 1.74-fold which is below the Guideline criteria of ≥ 2-fold; however, when the bac kground is subtracted, 10 μM forskolin induced testosterone an average of 2.7-fold. Es tradiol production was induced with 10 μ M forskolin by 9.6-fold. Compared to SC , 1 μM prochloraz on average inhibited the synthesis of testosterone 0.48-fold and estradiol to 0.35-fold. Thus, the guideline requirements were met indicating that the assa y was sensitive to induc tion and inhibition of testosterone and estradiol.

The %CVs based on absolute hormone concentr ation in SC well repl icates were 2.19-2.64% for testosterone and 1.89-4.01% for estradiol. The between-plate variability (calculated by the reviewer) for the QC plates based on the ab solute hormone concentrations in the SC was 18.8% for testosterone and 14.2% for estradiol. These data met the performance criteria recommended by the Guideline ( ≤ 30%).

TABLE 5. Mean (±SD) Hormone Concentrations Followi ng Treatment with Forskolin or Prochloraz for 48 Hours. a
Concentration (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b Testosterone (pg/mL) Fold Difference (Relative to DMSO) Background 1493 1035 1359 0.57 0.58 0.59 0.58 0.01 Blank 2921 1799 2338 1.12 1.01 1.01 1.05 0.06 DMSO 2612 1782 2319 1.00 1.00 1.00 1.00 -- 1 μM Forskolin 4325 2403 3108 1.66 c 1.35 c 1.34 c 1.45 c 0.18 10 μM Forskolin 5604 2561 3765 2.15 d 1.44 d 1.62 d 1.74 d 0.37 0.1 μM Prochloraz 2145 1505 1843 0.82 0.84 0.79 0.82 0.03 1 μM Prochloraz 1351 740 1168 0.52 0.42 0.50 0.48 0.05 Estradiol (pg/mL) Fold Difference (Relative to DMSO) Background 66 57 76 0.29 0.33 0.34 0.32 0.03 Blank 244 179 242 1.07 1.02 1.08 1.06 0.03 DMSO 229 175 223 1.00 1.00 1.00 1.00 -- 1 μM Forskolin 1550 1014 1559 6.76 5.79 6.99 6.51 0.64 10 μM Forskolin 2396 1363 2321 10.46 7.78 10.41 9.55 1.53 0.1 μM Prochloraz 168 151 176 0.73 0.86 0.79 0.79 0.07 1 μM Prochloraz 77 62 78 0.34 0.36 0.35 0.35 0.01 a Data were obtained from pages 21–22 of the study report. The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol. b Calculated by the reviewers. c When the background is subtracted, the valu es are 2.53-, 1.83-, and 1.82-fold difference for Runs 2, 3, and 4, respectively, with a mean of 2.06-fold change. d When the background is subtracted, the valu es are 3.67-, 2.04-, and 2.51-fold difference for Runs 2, 3, and 4, respectively, with a mean of 2.74-fold change.Steroidogenesis Assay (2011) / Page 14 of 14 EPTC / 041401 OCSPP 890.1550/ OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: EPTC was tested at a soluble concentration range (0.0001 to 100 μ M) and was determined not to affect test osterone or inhibit estradiol in this steroidogenesis assay in the H295R cell line. EPTC was associated with an induction of estradiol at 100 uM in the H295R cell line.

B. AGENCY COMMENTS: All guideline acceptability r ecommendations and requirements were met, including lack of cytotoxicity, adeq uate production of testos terone and estradiol, acceptable reproducibility (low %CV), and a ppropriate induction and inhibition with positive controls.

Testosterone concentration was not affected by EPTC. In three independent runs of the assay, statistically significant inhibition or induction of te stosterone production was not observed. Decreases (p ≤ 0.05) of 9–10% in estradiol we re observed at the 0.01, 0.001, and 0.0001 μM doses in Run 3. However, when the estradiol results in Run 3 were normalized to percent viability, the inhibition of estradiol at 0.0001, 0.001, and 0.01 μM was not statistically significant. Thes e decreases were minor and no t reproduced in the other two runs. Increases (p ≤ 0.05) of 10–15% in estradiol was obser ved at the 100 μM dose in Run 3 (with and without normalization to viability) and in Run 4. Although the effect was slight, this effect was statistically significant in two of three runs and was repr oducible. Therefore, it is concluded that EPTC may be associated with an induction of estradiol at 100 μM.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 Stability of EPTC in the test system was not reported.

 Total number of cell passages was 12-13, exceeding 10.

 The laboratory proficiency test data were not provided.

 Mean±SD and %CV for the fold-differences we re not reported, but could be calculated from the data presented.

# DATA EVALUATION RECORD

EPTC
Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-13-2012 (MRID 48616407)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Rebecca Bryan, B.S. Date: 1/11/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 1/25/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 1/27/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 1/27/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).EPTC/ 041401


### Primary Reviewer: Sheila Healy, Ph

. D

. Health Effects Division In vivo Uterotrophic Assay (2011) I Page I of8 OCSPP 890.1600 / OECD 440



### Secondary Reviewer: _J_e_s_s_R_o_w_l_a_nd

_________ Signature:**-~~~==-::.L_,:::.__** 

### Health Effects Division Date:**---~~---** Template version 09 / 2011 

### DAT A EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600 ; OECD 440

PC CODE: 041401 DP BARCODE: D397 l 77

TXR#: 0051762 CAS#: 759-94-4

TEST MATERIAL (PURITY): E PTC ( > 99.9% a.i.)

SYNONYMS: S -ethyl N,N -dipropylcarbamothioate

CITATION: Zorrilla, L. (20 11) The Uterotrophic Assay with E PTC in Ovarectomized CRL:CD(SD) IGS Rats

. Integrated Laboratory Systems, Inc
., Durham
, NC. La boratory Project Study ID : C201-100
, November 3, 2011. MRID 48616407

. Unp ublish ed

.

SPONSOR: Gowan Company
, P .O. Box 5569
, Yuma
, AZ

TEST ORDER#: E DSP-041401-64

EXECUTIVE SUMMARY: In an uterotrophic assay (MRID 48616407) conducted to screen for potential estroge nic activity, EPTC ( > 99.9% a.i., Lot# P 1105096) in corn oil was administere d daily via oral gavage to groups of eight ovariectomized female Sprague-Dawley
rats at dose le ve l s of 0 (vehicle), 50, or 200 mg / kg / day on post-natal days (PND) 59-61. A positive control group was treated w ith l 7a.-ethynyl estradiol (EE) by daily oral gavage at a dose level of 0.1 mg / kg / day. All animals were terminated and necropsied approximately 24 hour s after the final dose ( PND 62) to determin e wet and blotted uterine weights.

All animals survived until sc h e duled termination. No clinical signs of toxicity were observed in any of the EPTC treated gro up s. In the po sit ive control gro up (EE)
, one animal exhibited alopecia on the n eck on Da y 3
, which was considered incidental. No treatment-related effects on body weight or body weight ga ins were noted in the EPTC treated groups. In the positive control group (EE), an overall (Days 1-4) body weight loss of

- 0 .2 g (vs 

### + 12

. 6 g controls) was observed. Uterine weig ht s in the EPTC treated groups were comparable to the controls. Absolute wet an d blotted uterus we i g ht s for the EE group were increased (p :S 0.05) b y 89 % and 96%, respecti ve l y.

No statistica ll y sign ificant changes were seen in uterine weight in this assay. EPTC is negative in the uterotrophic assay.In vivo Uterotrophic Assay (2011) / Page 2 of 8 EPTC/ 041401 OCSPP 890.1600/ OECD 440



### The assay satisfies the EDSP Tier 1 Test Orde r requirements for a ut erotrophic assay (OCSPP 890.1600).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.In vivo Uterotrophic Assay (2011) / Page 3 of 8 EPTC/ 041401 OCSPP 890.1600/ OECD 440



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratory Systems, Inc.
Location: Durham, NC Study Director: L. Zorrilla Other Personnel: S. Borghoff (Study Toxicologist), P. Sproul (Toxicology Study Manager), A. Glasscock (Ani mal Facility Operations Manager), T. Hackett (Necropsy Manager), C. Swartz (Facility Veternarian), C. Cachafeiro (Health and Safety Officer), P. Reibach (Principal Investigator, Smithers Viscient). Study Period: May 8-11, 2011



### 2. Test Substance: EPTC Description: Off white solid Source: Gowan Company (commerc ial technical product) Lot #: P1105096, expires February 2, 2015 Purity: >99.9 % a.i. Stability: Stable in the vehicle for up to 25 days at 1-10°C (Smithers Viscient Inc., Study No. 12791-6162) CAS #: 759-94-4 Structure:

S N CH 3

CH 3 O

CH

3



### 3. Reference Estrogen: 17 α -ethynyl estradiol (EE) Supplier: Sigma Aldrich, St. Louis, MO Lot/Batch #: 090M1241V, expires February 2012 Purity: ≥ 98 % CAS # : 57-63-6



### 4. Solvent/Vehicle Control: Corn Oil Supplier: MP Biomedicals, LLC, Solon, OH Lot #: 7862K Rationale (if other than water): Not Provided Final concentration : Not Applicable



### 5. Test Animals: Species: Rat (ovariectomized females) Strain: Sprague-Dawley (Crl:CD[SD] IGS) Age/weight at dose initiation: PND 59 / 221.1-274.3 g females Source: Charles River Laboratories International, Inc., Raleigh, NC Housing: 2/cage in polycarbonate cages wi th absorbent heat-treated hardwood bedding (Northeastern Products Corp., Warrensburg, NY) Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum ; low phytoestrogen with total geinstein equivalents = 6.2 μg/g diet. Water: Reverse osmosis-treated tap water, ad libitumIn vivo Uterotrophic Assay (2011) / Page 4 of 8 EPTC/ 041401 OCSPP 890.1600/ OECD 440

Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 23-25oC 34-50% Not provided 12 hrs light/12 hrs dark Acclimation period: 10 days



### B. METHODS

1. In Life Dates: Start: May 8, 2011 End: May 11, 2011

2. Study Design: Following a 10-day acclimation period, ovariectomized female rats (surgery performed by Supplier) were administered the test substances for three consecutive days starting at PND 59. Rats were euthanized approximately 24 hours after the last dose and necropsied for uterine weight measurements.

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical anal ysis indicated that th ere were no significant differences between group mean weights at study initiation.

TABLE 1. Study Design of Estrogen Agonist Assay a Test Group Dose (mg/kg/day) # of Females Vehicle Control 0 8 Low EPTC 50 8 High EPTC 200 8 17 α -ethynyl estradiol (EE), Reference Estrogen 0.1 8 a Data were obtained from Table 1 on page 15 of the study report.



### 4. Dose Selection Rationale: The EPTC dose levels were se lected based on th e results from two developmental toxicity studies (study numbers not provided). In a range-finding developmental toxicity study, EPTC in corn o il was administered by da ily oral gavage to female COBS SD rats at dose levels of 50, 90, 160, 280, or 500 mg/kg/ day from gestation day (GD) 6 through 15. Mortality was obs erved at 500 mg/kg/day. At 160 and 280 mg/kg/day, body weight losses we re noted during GD 6-9, food consumption was reduced, and clinical signs of toxic ity (yellow urogenital matting and staining and red matter around the nose) were observed. In the definitive developmental study, EPTC was administered as before at dose levels of 30, 100, and 300 mg/ kg/day. Body weight losses and decreased food consumption were noted at 300 mg/kg day [S tauffer Chemical Company (1983) as reference in study report, not a MRID study 

### ] Based on these results, doses of 50 and 200 mg/kg/day were selected for the curr ent study. For the positive control, 17 α -ethynyl estradiol (EE), a dose of 0.1 mg/kg/day was se lected based on the previous experience within the testing laborator y as described in a baselin e positive control study.

5. Dose Preparation and Analysis: Dose formulations were pr epared once prior to initiation of treatment by suspending the appropriate amounts of test substance in corn oil

. TheIn vivo Uterotrophic Assay (2011) / Page 5 of 8 EPTC/ 041401 OCSPP 890.1600/ OECD 440


### reference estrogen, EE, was mixed with corn oil for dosing. Doses were adjusted daily based on individual body weight measurements. Homogeneity and concentration analyses were measured prior to administration in th e 10 and 40 mg a.i./mL formulations. In a previous storage stability study 1 

### , EPTC (lot number not report ed) was determined to be stable in corn oil for up to 25 days at 1-10°C.

Results of Dose Analysis

Homogeneity (% CV): 1.66-4.47%

Stability (% of Day 0): Data not provided.

Concentration (% of nominal): 93-96% (The initial low dose analysis was slightly out of range at 84.9% of nominal, therefore the low dose formulation used in the current study is the same low dose formulation as the c oncurrent EPTC Hers hberger assay, MRID 48616406).

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: Animals were administered the test formulations, EE, or vehicle daily via oral gavage for three consecutive days in a dose volume of 5 mL/kg body weight. Dose volumes were adjusted daily based on the concurrent body weight measurement.

7. Statistics: Final body weights, body we ight gains, and uterine we ights were analyzed using studentized residual plots for outliers and Levene ’s test for homogeneity of variance. Data were analyzed using one-way ANOVA followed by Dunnett’s one tailed t-test (uterine weights) and Dunnett’s two tailed t-test (f inal body weight and body weight gain). Significance was denoted at p ≤ 0.05. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Animals were checked for mort ality or moribundity twice daily on weekdays and once daily on weekends and holiday s. Rats were also checked for clinical signs of toxicity prior to dose administra tion and three hours (±30 min) post-dosing.

2. Body Weight: Animals were weighed at randomiza tion, daily prior to dosing throughout the dosing period, and at termination.

3. Food Consumption (Optional): Food consumption was not measured.

1 Reibach, R. (2011) Storage Stability of EPTC in Corn Oil. Unpublished study re port prepared by Smithers Viscient Inc., Study No. 12791-6162.In vivo Uterotrophic Assay (2011) / Page 6 of 8 EPTC/ 041401 OCSPP 890.1600/ OECD 440


### 4. Necropsy and Measurement of Uterine Weight: Approximately 24 hours after final administration of the test substance, all surviving animals were euthanized by CO 2 

### inhalation followed by cervical dislocation and subjected to a gross ne cropsy. The uterus and vagina were removed and excess fat and connective tissue were trimmed. The vagina was removed from the uterus below the cervix, and the uterus was weighed (to obtain a wet weight). The uterus was then pierced and blotted to remove luminal fluid, and weighed again (to obtain a blotted weight).

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived unt il scheduled termination.

2. Clinical Signs of Toxicity: No clinical signs of toxicity were observed in any of the EPTC treated groups. In the positive control group (EE), one animal exhibited alopecia on the neck on Day 3, which was considered incidental.

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are presented in Table 2. No treatment-relate d effects on body weight or body weight gains were noted in the EPTC treated groups. In the positive control group (EE), an overall (Days 1-4) body weight loss (p ≤ 0.05) of

− 0.2 g (vs +12.6 g controls) was observed.

TABLE 2. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

Study Day # Dose (mg/kg/day) Vehicle Control EPTC (50) EPTC (200) EE (0.1)

N Mean SD N Mean SD N Mean SD N Mean SD 1 8 245.5 16.3 8 247.9 13.7 8 246.7 13.4 8 248.5 11.8 2 8 244.8 14.7 8 247.3 14.3 8 245.7 15.0 8 246.5 13.9 3 8 249.3 14.4 8 254.2 14.6 8 250.7 15.3 8 245.3 12.6 4 8 258.0 15.9 8 261.0 15.5 8 257.9 16.1 8 248.3 12.7 Body Weight Gain (Days 1-4) 8 12.6 2.4 8 13.1 2.7 8 11.2 5.1 8 -0.2* 3.1

a Data were obtained from Table 3 on page 19 and Appendix V on page 51 of the study report. Percent differences from controls were calculated by the re viewers and included in parentheses. N= No. of animals in the group SD = Standard Deviation * Significantly different from controls at p ≤ 0.05.In vivo Uterotrophic Assay (2011) / Page 7 of 8 EPTC/ 041401 OCSPP 890.1600/ OECD 440


### C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. PATHOLOGY

1. Uterine Weights: Uterine weight data are presented in Table 3. Uterine weights in the EPTC treated groups were comparable to the controls. The mean bl otted vehicle control uterine weight was 0.025% of the mean vehicl e control terminal body weight, which meets the Guideline requirement (<0.04%). Absolute wet and blotted uterus weig hts for the EE group were increased (p ≤ 0.05) by 89% and 96%, respectively. These in creased uterine weights were consistent with an estrogen receptor agonist.

TABLE 3. Uterine Weights from Es trogen Agonist Assay in SD Rats a

Parameter Dose (mg/kg/day) Vehicle Control EPTC (50) EPTC (200) EE (0.1) N Mean SD N Mean SD N Mean SD N Mean SD Terminal BW (g) 8 258.0 15.9 8 261.0 15.5 8 257.9 16.1 8 248.3 12.7 Wet, absolute (mg) 8 78.3 16.9 8 80.2 16.1 7 68.1 21.9 8 148.1* ( ↑ 89) 24.5

Wet, relative (%) b 8 0.030 0.007 8 0.031 0.007 8 0.026 0.008 8 0.060 0.013 Blotted, absolute (mg) 8 64.1 10.9 8 59.6 14.7 8 56.6 15.2 8 125.4* ( ↑ 96) 24.6

Blotted, relative (%) b 8 0.025 0.005 8 0.023 0.006 8 0.022 0.005 8 0.051 0.012 a Data were obtained from Table 4 on page 20 and Appendix VI on page 53 of the study report. b Relative wet and blotted uterine weights were calculated by the reviewer from the individual data. BW= body weight N= No. of animals in the group SD = Standard Deviation * Significantly different from vehicle control at p ≤ 0.05.



### 2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: There were no clinical si gns observed that were attributed to administration with EPTC or refe rence substance. EPTC administered up to 200 mg/kg did not result in a change in body weight, body weight gain or uterine weight, wet or blotted, as compared to the vehicle control group. The positive control 17 α -ethinyl estradiol, a known estrogen agonist, induced a statistically significant increase in both wet and blotted uterine weights compared to the vehicle control group. Or al administration of EPTC up to 200 mg/kg/day for three consecutive days to ovariectomized adult rats did not result in an estrogenic response as shown by a lack of effect on uterine weights.In vivo Uterotrophic Assay (2011) / Page 8 of 8 EPTC/ 041401 OCSPP 890.1600/ OECD 440



### B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical signs of toxicity were observed in any of the EPTC treated groups. In the positive control group (EE), one animal on Day 3 exhibited al opecia on the neck which was considered incidental. No treatment-relate d effects on body weight or body weight gains were noted in the EPTC treated groups. In the positive co ntrol group (EE), an ove rall body weight loss (p ≤ 0.05) of

− 0.2 g (vs. +12.6 g controls) was observed. Uterine weights in the EPTC treated groups were comparable to the controls. Abso lute wet and blotted uterus weights for the EE group were increased (p ≤ 0.05) by 89% and 96%, respectively. No statistically significant changes were seen in uterine weight in this assay. EPTC is negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: None